Method of Producing Rough Strains of Bacteria and Uses Thereof

ABSTRACT

The present invention relates to a method of producing rough strains of a bacterium, such as  Mycobacterium obuense , said method comprising exposing said bacterium to a sulfone and/or sulfonamide (such as 4,4′-Diaminodiphenyl sulfone or an analogue thereof). A rough strain of  Mycobacterium obuense  producible by said method and uses thereof. In particular, uses of a rough strain of  Mycobacterium obuense  deposited under the Budapest Treaty of NCTC with the accession number NCTC 13365.

FIELD OF THE INVENTION

The present invention relates to rough strains of bacteria, such asthose from the genus Mycobacterium. In particular, the present inventionrelates to methods of obtaining rough strains of bacteria. The presentinvention yet further relates to a novel Mycobacterium obuense roughstrain and use of this rough strain of Mycobacterium obuense.

BACKGROUND TO THE INVENTION

Methods of obtaining rough strains of bacteria, such as those from thegenus Mycobacterium, that are known in the art include the plating outof bacteria, such as those from the genus Mycobacterium, and looking forany rough colonies. However, any rough strains identified in this wayare typically not stable.

Other methods include plating out Mycobacteria on medium that containsTween or glycerol and encouraging pellicle growth. This method has beensuccessful in inducing rough colonies of some species of Mycobacteria.However, this method has not been able to induce rough variants in allMycobacteria.

No method to date has been able to induce rough strains of Mycobacteriumobuense.

Accordingly, there is a need for an improved method that is able toproduce rough strains, particularly stable rough strains, ofMycobacterium. Such a method could advantageously be used to produce forthe first time rough strains of some species of Mycobacterium such asMycobacterium obuense.

Advantageously, the present invention has provided such an improvedmethod that has induced for the first time a rough strain, particularlya stable rough strain, of Mycobacterium obuense.

Rough strains of fast-growing Mycobacteria have a number of advantages:

-   -   stable rough strains do not revert to smooth strains;    -   rough strains have a different presentation of antigens and tend        to be less virulent in causing disease than smooth strains;    -   skin test reagents made from rough strains are more effective        than those made from smooth strains;    -   rough strains are very effective when used in in vitro        cell-mediated immune experiments; and    -   the small clumps or particles of several bacilli may be more        effective in stimulating phagocytic cells than single organisms        of smooth strains.

Furthermore, the present invention has advantageously found that a wholecell of a rough strain of Mycobacterium obuense may be used to modulatethe immune response of a subject.

Although M. vaccae was known to modulate the immune response of asubject—M. obuense had never before been considered suitable for use asan immune modulator.

Vaccines and other immune modulators have a major impact in reducingmorbidity and mortality from disease. The primary immunity elicited bymost current vaccines appears to be mediated by the humoral immuneresponse. For diseases that may require a cellular immune response, suchas tuberculosis and leishmaniasis, there are currently no availablevaccines that are uniformly effective.

Typically, adjuvants are added to vaccines. The role of the adjuvant isto enhance the body's immune response to specific antigens of thevaccine. Commonly used adjuvants typically produce a humoral immuneresponse but not a cell-mediated immune response. In addition, aluminiumadjuvants for example may cause negative side effects, such as sterileabscesses, erythema, swelling, subcutaneous nodules, granulomatousinflammation and contact hypersensitivity.

A vaccine or other immune modulator is sought that modifies a cellularimmune response and in particular the T helper cell response, forexample, the T helper cell 1 (Th1) and T helper cell 2 (Th2) response.

There are many different autoimmune diseases, and they can each affectthe body in different ways. Many of the autoimmune diseases are rare. Asa group, however, autoimmune diseases afflict millions of people.

Some autoimmune diseases are known to begin or worsen with certaintriggers such as viral, parasitic and chronic bacterial infections.Other less understood influences affect the immune system and the courseof autoimmune diseases include ageing, chronic stress, hormones andpregnancy.

Autoimmune diseases are often chronic, requiring lifelong care andmonitoring, even when the person may look or feel well. Currently, fewautoimmune diseases can be cured or made to go into remission withtreatment.

Physicians most often help patients manage the consequences ofinflammation caused by the autoimmune disease. In some people, a limitednumber of immuno-suppressive medications may result in diseaseremission. However, even if their disease goes into remission, patientsare rarely able to discontinue medication. The long-term side effects ofimmunosuppressive medication can be substantial.

Initiation and progression of vascular injury is a complex,multi-factorial process, but there is growing evidence that inflammatoryresponses play a key role. Vascular injury is involved in thedevelopment of atherosclerosis, and in thrombotic processes that lead toacute ischaemic syndromes such as myocardial infarction, stroke andperipheral artery occlusion.

Immune mechanisms may be important in the development and maintenance ofatherosclerosis and myointimal hyperplasia (MIH).

Myointimal Hyperplasia (MIH) can be considered as an exaggerated healingresponse to injury such as balloon angioplasty. A cascade of eventsresults in: loss of the basement membrane, migration of vascular smoothmuscle cells (VSMC) from the media into the intima, VSMC proliferationand phenotypic change to a more secretory fibroblastic cell type andincreased production of extracellular matrix, which eventually leads tostenosis or occlusion of the vessel. It occurs after bypass grafting andballoon angioplasty and affects approximately 30% of such cases inclinical practice. It is the major cause of failure of such proceduresand treatment of the resulting stenosed and blocked vessels/grafts isproblematic. The underlying cellular mechanisms leading to MIH are notwell understood and to date no therapy had been developed which caneffectively prevent it. The clinical relevance of the current patentrelates to the very large numbers of coronary artery angioplasties whichare performed annually in the UK and world-wide. Although drug elutingstents are currently producing promising results they are unlikely toprevent restenosis completely. Any safe, relatively inexpensiveadjunctive therapy, such as the immunotherapy proposed in this patent,would have a major clinical impact.

The mechanisms involved in immunotherapy against restenosis are complexand not completely elucidated. The endothelial injury caused byangioplasty may be exacerbated by the host immune response to hsp's.Hsp's are proteins produced by stressed cells which have been implicatedin the pathogenesis and the pathophysiology of various immunologicaldisorders including atherosclerosis (Xu Q et al. Arterioscler Thromb1992; 12: 789-799). It is likely that they will be present onendothelial and smooth muscle cells in the region of an angioplasty. Ineffect the hsp acts as an autoantigen which can then be attacked by theimmune system. This situation can be induced experimentally byimmunising with a cross-reactive mycobacterial hsp (hsp65) which leadsto endothelial damage in rabbits and mice (Xu Q, et al. ArteriosclerThromb 1992; 12: 789-799 and George J, et al. Circ. Res. 2000; 86:1203-1210). The effect appears to be dependent on IL-4 secreted by Th2lymphocytes, and is probably mediated by antibody George J, et al. Circ.Res. 2000; 86: 1203-1210 and Schett G, et al. J. Clin. Invest. 1995; 96:2569-2577). The relevance of these observations to man is suggested bythe ability of affinity-purified human antibody eluted from hsp65columns to damage stressed human endothelial cells in vitro. Thisfinding suggests that the antibody cross-reacts with hsp60 which is thehuman homologue of hsp65, and may be accessible to antibody whenexpressed on the membranes of stressed endothelial cells. It has beensuggested that such antibodies binding to stressed endothelial cells maybe a factor in producing coronary artery disease after hearttransplantation (Crisp S J et al. J Heart Lung Transplant 1994; 81-91).Mukherjee et al (Thromb Haemost 1996; 75: 258-60) showed no associationbetween preoperative antibody levels to hsp65 and coronary restenosis,but did show that those patients where levels of such antibodies droppedafter angioplasty were less likely to restenose. In fact the role ofantibodies to hsp could be complex, because patients with vasculardisease have not only raised antibody, but also raised levels of the hspthemselves (Wright B H, et al Heart Vessels 2000; 15: 18-22). Thus anapparent fall in antibody levels may merely reflect an increase inlevels of the protein. Moreover the hsp have regulatory effects, andbind to arterial smooth muscle cells, leading to enhanced survivalwithout a requirement for internalisation (Johnson A D et al.Atherosclerosis 1990; 84: 111-119).

WO2004/022093 and UK application number 0404102.6 (both of whichreferences are incorporated herein by reference) disclose an immunemodulator composition or a pharmaceutical composition comprising a wholecell of a bacterium from the genera Rhodococcus, Gordonia, Nocardia,Dietzia, Tsukanmurella and Nocardioides.

In addition, U.S. application Ser. No. 10/893,524 (incorporated hereinby reference) discloses the use of a whole cell of a bacterium from thegenera Rhodococcus, Gordonia, Nocardia, Dietzia, Tsukamurella andNocardioides in a manufacture of a medicament for the treatment orprevention of post-weaning multisystemic wasting syndrome (PMWS) and/orporcine dermatitis and nephropathy syndrome (PDNS).

However, none of these documents teaches or suggests the use of wholecells of rough strain, preferably a stable rough strain, of M. obuenseto modulate a cellular immune response.

SUMMARY OF THE INVENTION

The present invention is predicated upon the surprising finding that arough strain of a bacterium, such as a rough strain of a bacterium fromthe genus Mycobacterium, may be induced by exposure to a sulfone and/ora sulfonamide.

From birth to death the immune system is educated, constantly stimulatedand regulated through contact with the environment. Modern urbanisationand public health measures to prevent infectious disease have virtuallyeliminated this exposure leading to an unprecedented rise indiseases—such as allergies and neoplastic diseases. Restoring thebeneficial affects of the environment through the use of killedsuspensions of harmless beneficial environmental bacteria may redressthe normal balance of the immune system thus acting, therapeuticallyand/or prophylactically in the treatment of diseases and/or in promotinga healthy immune system.

Thus, in addition or in the alternative, to the method of inducing roughstrains of bacteria (such as Mycobacterium e.g. M. obuense), the presentinvention is predicated upon the surprising finding that a whole cell ofa bacterium from a rough strain of Mycobacterium obuense administered toa test subject is particularly effective at eliciting a modification ofthe immune system, in particular the cellular immune system, of thattest subject.

The phrase “cellular immune system”, as used herein, includes acell-mediated immune response which depends upon the presence of Tlymphocytes. The term “T lymphocytes” includes cytotoxic T lymphocytes,helper T cells, suppresser T cells and regulatory T cells. Modificationof a cell-mediated immune response may be used, for example, to overcomecell-mediated immune disorders including for example an immune systemimbalance and immune hypersensitivity.

The terms “modulate”, “modify”, “modification” and other derivativesthereof, as used herein, mean downregulating, inhibiting, inducing,stimulating, upregulating, altering or otherwise affecting a componentor components of the cellular immune system.

The present invention is predicated upon the surprising finding that awhole cell of a rough strain of Mycobacterium obuense administered to atest subject is particularly effective at eliciting a modification ofthe immune system, in particular the cellular immune system of that testsubject, which effects a preventative and/or therapeutic effect onautoimmune diseases or autoimmune disorders, particularly those whichinvolve the inflammation of the intima of blood vessels for example.

An advantage of the use of compositions comprising a whole cell of abacterium from a rough strain of M. obuense to effectively treat and/orprevent autoimmune diseases and autoimmune disorders, particularly thosewhich involve the inflammation of the intima of blood vessels forexample, may be that this treatment and/or prevention is effected whilstproducing fewer long-term side effects than the chemotherapies, i.e. theimmunosuppressive medication, now routinely used.

DETAILED ASPECTS OF THE PRESENT INVENTION

In one aspect, the present invention relates to a method of producing arough strain of a bacterium, said method comprising exposing saidbacterium to a sulfone and/or a sulfonamide

Suitably, in a method of producing rough strains of a bacteriumaccording to the present invention the rough strain may be isolated.

Preferably, in a method of producing rough strains of a bacteriumaccording to the present invention a bacterium is grown on medium asulfone and/or a sulfonamide at a concentration of greater than or equalto 5 μg sulfone and/or sulfonamide per 1 ml medium.

Suitably, in a method of producing rough strains of a bacteriumaccording to the present invention said bacterium may be from the genusMycobacterium. Preferably from Mycobacterium obuense.

In another aspect, the present invention relates to a rough strain of abacterium from the genus Mycobacterium producible, preferably produced,by a method of the present invention.

In a further aspect, the present invention relates to a rough strain ofMycobacterium obuense producible, preferably produced, by a method ofthe present invention.

In another aspect, the present invention relates to a rough strain ofMycobacterium obuense that has been deposited by BioEos Limited of 67Lakers Rise, Woodmansterne, Surrey, SM7 3LA under the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thepurposes of Patent Procedure at the National Collection of Type Cultures(NCTC), Central Public Health Laboratory, 61 Colindale Avenue, London,NW9 5HT), under Accession Number NCTC 13365 on the 14 Jul. 2005.

In one aspect, the present invention provides an immune modulatorcomposition comprising a whole cell of a rough strain of Mycobacteriumobuense.

The term “immune modulator”, as used herein, means a substance whichmodulates a cellular immune system of a subject.

The term “whole cell”, as used herein, means a bacterium which isintact, or substantially intact. In particular, the term “intact” asused herein means a bacterium which is comprised of all of thecomponents present in a whole cell, particularly a whole, viable cell,and/or a bacterium which has not been specifically treated to remove oneor more components from it. By the term “substantially intact” as usedherein it is meant that although the isolation and/or purificationprocess used in obtaining the bacterium may result in, for example, aslight modification to the cell and/or in the removal of one or more ofthe components of the cell, the degree to which such a modificationand/or removal occurs is insignificant. In particular, a substantiallyintact cell according to the present invention has not been specificallytreated to remove one or more components from it.

Prior to the present invention the use of whole cells of bacterium froma rough strain of Mycobacterium obuense to modulate a cellular immuneresponse was not contemplated. Surprisingly, it has been found that byusing a whole cell of a rough strain of Mycobacterium obuense,modulation of a cellular immune system can be effected. The modulationof a cellular immune response caused by administration of said wholecell of said rough strain may be advantageously long lasting as comparedwith the response elicited by administration of an individual componentof the bacterium.

Preferably, the composition according to the present invention comprisesmore than one whole cell, and more preferably comprises a plurality ofwhole cells.

In a further aspect, the present invention provides an immune modulatorcomposition comprising a whole cell of a bacterium from a rough strainof Mycobacterium obuense, which immune modulator composition in usemodifies a cellular immune response.

In another aspect, the present invention provides an immune modulatorcomposition comprising an antigen and an adjuvant, wherein said adjuvantcomprises a whole cell of a bacterium from a rough strain ofMycobacterium obuense

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a whole cell of a bacterium from a rough strainof Mycobacterium obuense and optionally a pharmaceutically acceptablecarrier, diluent or excipient, which immune modulator composition in usemodifies a cellular immune response.

The present invention yet further provides a process of preparation of apharmaceutical composition of the present invention, said processcomprising admixing one or more of the compounds of the presentinvention with a pharmaceutically acceptable diluent, excipient orcarrier.

In a further aspect, the present invention provides an immune modulatorcomposition and/or a pharmaceutical composition comprising a whole cellof a bacterium from a rough strain of Mycobacterium obuense and at leastone antigen or antigenic determinant.

Suitably, the antigen or antigenic determinant may be an antigen orantigenic determinant from one or more of the following: BCG (bacillusof Calmette and Guerin) vaccine, diphtheria toxoid vaccine,diphtheria/tetanus/pertussis (DTP or Triple) vaccine, pertussis vaccine,tetanus toxoid vaccine, measles vaccine, mumps vaccine, rubella vaccine,OPV (oral poliomyelitis vaccine), Mycobacterium vaccae, or part thereof(as taught in GB0025694.1) and a generic plasmodium antigen, for examplea malaria parasite antigen.

Suitably, the immune modulator composition and/or pharmaceuticalcomposition may comprise two or more such antigens or antigenicdeterminants.

In another aspect, the present invention provides an immune modulatorcomposition comprising an antigen or an antigenic determinant and anadjuvant, wherein said antigen or antigenic determinant comprises awhole cell of a bacterium from a rough strain of Mycobacterium obuense.

When it is the case that whole cell of the bacterium functions as anantigen or antigenic determinant the composition may suitably compriseat least one, preferably at least two, more preferably at least three,further antigens or antigenic determinants.

The present invention further provides the use of an immune modulatorcomposition or a pharmaceutical composition comprising a whole cell of abacterium from a rough strain of Mycobacterium obuense, in themanufacture of a medicament for the treatment or prevention of one ormore of: an infection (e.g. a bacterial, viral, for example an infectioncaused by papilloma viruses, including equine sarcoid, genital warts anddysplasia of the uterine cervix that precedes carcinoma of the cervix,or parasitic infection, for example, malaria, trypanosomiasis,leishmaniasis, infection with Eimeria species in poultry andtoxoplasmosis) and/or the immunological abnormalities accompanying aninfection; an autoimmune disease (e.g. a vascular disorder, such asobliterative vascular disorder, and the immunological aspects underlyingmyointimal hyperplasia and/or atheroma formation (otherwise known asarteriosclerosis), arthritis and graft rejection.); stress (for example,major trauma stress, psychosocial stress and chronic stress); an allergy(e.g. asthma including allergic asthma, hayfever, allergic dermatitis(eczema), anaphylactic shock, allergies to plant contact or ingestion,stings—such as nettle and insect stings, and allergies to insectbites—such as midges, for instance Culicoides(which causes Sweet Itch inhorses)); heaves; COPD; PMWS; PDNS; SIPH; cancer (for example melanomaor adenocarcinoma); an immune system imbalance (e.g. an immune systemimbalance in children and the elderly); and post-operative stress andinfection. An immune system imbalance in the elderly may be referred toas immunosenescence.

The present invention further provides the use of an immune modulatorcomposition or a pharmaceutical composition comprising a whole cell of abacterium from a rough strain of Mycobacterium obuense, in themanufacture of a medicament for the treatment or prevention of one ormore viral infections, for example an infection caused by papillomaviruses, including equine sarcoid, genital warts and dysplasia of theuterine cervix that precedes carcinoma of the cervix.

In one aspect, the present invention provides an immune modulatorcomposition and/or a pharmaceutical composition comprising a whole cellof a bacterium from a rough strain of Mycobacterium obuense, and atleast one antigen or antigenic determinant, wherein said antigen orantigenic determinant is a viral antigen of bovine papilloma viruses.

The present invention further provides the use of an immune modulatorcomposition or a pharmaceutical composition comprising a whole cell of abacterium from a rough strain of Mycobacterium obuense, in themanufacture of a medicament for the treatment or prevention of one ormore of a parasitic infection, such as, for example, malaria,trypanosomiasis, leishmaniasis, infection with Eimeria species inpoultry and toxoplasmosis.

The present invention further provides the use of an immune modulatorcomposition or a pharmaceutical composition comprising a whole cell of abacterium from a rough strain of Mycobacterium obuense, in themanufacture of a medicament for the treatment or prevention of one ormore of an autoimmune disease, a vascular disorder, such as anobliterative vascular disorder and the immunological aspects underlyingmyointimal hyperplasia and/or atheroma formation (otherwise known asarteriosclerosis), arthritis and graft rejection.

The present invention further provides the use of an immune modulatorcomposition or a pharmaceutical composition comprising a whole cell of abacterium from a rough strain of Mycobacterium obuense, in themanufacture of a medicament for the treatment or prevention of stress,such as, for example, major trauma stress, psychosocial stress and/orchronic stress and/or post-operative stress (including the stressrelated to being administered anaesthetics).

The present invention further provides the use of an immune modulatorcomposition or a pharmaceutical composition comprising a whole cell of abacterium a rough strain of Mycobacterium obuense, in the manufacture ofa medicament for the treatment or prevention of one or more of asthma(including allergic asthma), hayfever, allergic dermatitis (eczema),anaphylactic shock, allergies to plant contact or ingestion, stings—suchas nettle and insect stings, and allergies to insect bites—such asmidges for instance Culicoides(which causes Sweet Itch in horses).

Preferably, the immune modulator composition or pharmaceuticalcomposition comprising a whole cell of a bacterium from a rough strainof Mycobacterium obuense is used in the manufacture of a medicament forthe treatment or prevention of asthma including allergic asthma, andallergies to insect bites—such as midges for instance Culicoides(whichcauses Sweet Itch in horses).

The present invention further provides the use of an immune modulatorcomposition or a pharmaceutical composition comprising a whole cell of abacterium from a rough strain of Mycobacterium obuense in themanufacture of a medicament for the treatment or prevention of one ormore of heaves and/or COPD, particularly in horses.

The present invention further provides the use of an immune modulatorcomposition or a pharmaceutical composition comprising a whole cell of abacterium a rough strain of Mycobacterium obuense, in the manufacture ofa medicament for the treatment or prevention of one or more of PMWSand/or PDNS.

The present invention further provides the use of an immune modulatorcomposition or a pharmaceutical composition comprising a whole cell of abacterium from a rough strain of Mycobacterium obuense, in themanufacture of a medicament for the treatment or prevention ofstress-induced pulmonary haemorrhage (SIPH), preferably exercise-inducedpulmonary haemorrhage (EIPH).

The present invention further provides the use of an immune modulatorcomposition or a pharmaceutical composition comprising a whole cell of abacterium a rough strain of Mycobacterium obuense, in the manufacture ofa medicament to for the treatment or prevention of melanoma and/oradenocarcinoma.

The present invention further provides the use of an immune modulatorcomposition or a pharmaceutical composition comprising a whole cell of abacterium a rough strain of Mycobacterium obuense, in the manufacture ofa medicament for the treatment or prevention of an immune systemimbalance in the elderly. Typically, an immune modulator composition ora pharmaceutical composition according to this aspect of the presentinvention may be an immune enhancer.

The present invention further provides the use of an immune modulatorcomposition or a pharmaceutical composition comprising a whole cell of abacterium from a rough strain of Mycobacterium obuense, in themanufacture of a medicament for enhancing the immune system which mayresult in for example, enhancement of growth or an increase in theefficiency of feed utilisation. Typically, the immune modulatorcomposition or pharmaceutical composition according to this aspect ofthe present invention may be an immune enhancer. Advantageously, theimmune modulator composition or pharmaceutical composition of thepresent invention may be used to replace antibiotics that are currentlyused to promote the growth of livestock. Suitably, the immune modulatorcomposition of the present invention may be used either alone or incombination with other treatments. The term “livestock”, as used hereinrefers to any farmed animal. Preferably, livestock is one or more ofhorses, poultry, pigs (including piglets), sheep (including lambs), cowsor bulls (including calves). More preferably, livestock meanspigs—including piglets.

The present invention further provides the use of an immune modulatorcomposition or a pharmaceutical composition comprising a whole cell of abacterium from a rough strain of Mycobacterium obuense, in themanufacture of a medicament for the treatment or prevention of an immunesystem imbalance in children.

The present invention further provides the use of an immune modulatorcomposition or a pharmaceutical composition comprising a whole cell of abacterium from a rough strain of Mycobacterium obuense, in themanufacture of a medicament for the treatment or prevention of anadverse reaction to childhood vaccines—such as whooping coughvaccinations and the current MMR vaccinations—and/or consequencesthereof. The term “adverse reaction”, as used herein, means a local orgeneralised disadvantageous response caused by or primed by the vaccineor the administration thereof, which typically occurs within a shorttime-frame but which can be delayed (for example by 6-months). An“adverse reaction” may include death of the child. The adverse reactionmay be caused as a consequence of a separate event, the response towhich has been negatively primed by the vaccine or the administrationthereof.

The present invention further provides the use of an immune modulatorcomposition or a pharmaceutical composition comprising a whole cell of abacterium a rough strain of Mycobacterium obuense in the manufacture ofa medicament for modifying a cellular immune response.

In a further aspect, the present invention provides an immune modulatorcomposition or a pharmaceutical composition comprising a whole cell of abacterium from a rough strain of Mycobacterium obuense for use as amedicament.

In a further aspect, the present invention provides an immune modulatorcomposition or a pharmaceutical composition comprising a whole cell of abacterium a rough strain of Mycobacterium obuense for use in or as avaccine.

Suitably, the vaccine may be a prophylactic vaccine or a therapeuticvaccine.

In a further aspect, the present invention provides an immune modulatorcomposition or a pharmaceutical composition comprising a whole cell of abacterium a rough strain of Mycobacterium obuense for use as an immuneenhancer.

In a further aspect, the present invention provides an immune modulatorcomposition or a pharmaceutical composition comprising a whole cell of arough strain of Mycobacterium obuense for use in the treatment orprevention of one or more of: an infection (e.g. a bacterial, viral, forexample an infection caused by papilloma viruses, including equinesarcoid, genital warts and dysplasia of the uterine cervix that precedescarcinoma of the cervix, or parasitic infection, for example, malaria,trypanosomiasis, leishmaniasis, infection with Eimeria species inpoultry and toxoplasmosis) and/or the immunological abnormalitiesaccompanying an infection; an autoimmune disease (e.g. a vasculardisorder, such as obliterative vascular disorder and the immunologicalaspects underlying myointimal hyperplasia and/or atheroma formation(otherwise known as arteriosclerosis), arthritis and graft rejection);stress (for example, major trauma stress, psychosocial stress andchronic stress); an allergy (e.g. asthma including allergic asthma,hayfever, allergic dermatitis (eczema), anaphylactic shock, allergies toplant contact or ingestion, stings—such as nettle and insect stings, andallergies to insect bites—such as midges, for instance Culicoides (whichcauses Sweet Itch in horses)); heaves; COPD; PMWS; PDNS; SIPH and cancer(for example melanoma or adenocarcinoma, or virally related cancers suchas cervical cancers for example); an immune system imbalance (e.g. animmune system imbalance in children and the elderly) and post-operativestress and post-operative infection.

In a further aspect, the present invention provides an immune modulatorcomposition or a pharmaceutical composition comprising a whole cell of abacterium a rough strain of Mycobacterium obuense for use in thetreatment or prevention of a parasitic infection, such as, for example,one or more of malaria, trypanosomiasis, leishmaniasis, infection withEimeria species in poultry and toxoplasmosis.

In a further aspect, the present invention provides an immune modulatorcomposition or a pharmaceutical composition comprising a whole cell of abacterium from a rough strain of Mycobacterium obuense for use in thetreatment or prevention of a viral infection, for example an infectioncaused by papilloma viruses, including equine sarcoid, genital warts anddysplasia of the uterine cervix that precedes carcinoma of the cervix.

In a further aspect, the present invention provides an immune modulatorcomposition or a pharmaceutical composition comprising a whole cell of abacterium from a rough strain of Mycobacterium obuense, for use in thetreatment or prevention of one or more of an autoimmune disease, avascular disorder, such as an obliterative vascular disorder and theimmunological aspects underlying myointimal hyperplasia and/or atheromaformation (otherwise known as arteriosclerosis), arthritis and graftrejection.

In a further aspect, the present invention provides an immune modulatorcomposition or a pharmaceutical composition comprising a whole cell of abacterium a rough strain of Mycobacterium obuense for use in thetreatment or prevention of stress, such as, for example, one or more ofmajor trauma stress, psychosocial stress and chronic stress, and/orpost-operative stress.

In a further aspect, the present invention provides an immune modulatorcomposition or a pharmaceutical composition comprising a whole cell of abacterium from a rough strain of Mycobacterium obuense for use in thetreatment or prevention of one or more of asthma (including allergicasthma), hayfever, allergic dermatitis (eczema), anaphylactic shock,allergies to plant contact or ingestion, stings—such as nettle andinsect stings, and allergies to insect bites—such as midges for instanceCulicoides (which causes Sweet Itch in horses). Preferably, the immunemodulator composition or a pharmaceutical composition comprising a wholecell of a bacterium from a rough strain of Mycobacterium obuense is foruse in the treatment or prevention of asthma, including for exampleallergic asthma, and allergies to insect bites—such as midges, forinstance Culicoides(which causes Sweet Itch in horses).

In a further aspect, the present invention provides an immune modulatorcomposition or a pharmaceutical composition comprising a whole cell of abacterium from a rough strain of Mycobacterium obuense for use in thetreatment or prevention of one or more of heaves and/or COPD,particularly in horses.

In a further aspect, the present invention provides an immune modulatorcomposition or a pharmaceutical composition comprising a whole cell of abacterium from a rough strain of Mycobacterium obuense for use in thetreatment or prevention of one or more of PMWS and/or PDNS, particularlyin pigs.

In a further aspect, the present invention provides an immune modulatorcomposition or a pharmaceutical composition comprising a whole cell of abacterium from a rough strain of Mycobacterium obuense for use in thetreatment or prevention of SIPH, particularly in fish (such as koi)and/or racing animals (such as horses, camels, greyhounds and humans).

In a further aspect, the present invention provides an immune modulatorcomposition or a pharmaceutical composition comprising a whole cell of abacterium from a rough strain of Mycobacterium obuense for use in thetreatment or prevention of cancer (for example melanoma and/oradenocarcinoma and/or virally related cancers such as cervical cancerfor example).

In a further aspect, the present invention provides an immune modulatorcomposition or a pharmaceutical composition comprising a whole cell of abacterium from a rough strain of Mycobacterium obuense for use in thetreatment or prevention of an immune system imbalance, particularlyimmunosenescence, in the elderly.

In a further aspect, the present invention provides an immune modulatorcomposition or a pharmaceutical composition comprising a whole cell of abacterium from a rough strain of Mycobacterium obuense for use in thetreatment or prevention of post-operative infection.

In a further aspect, the present invention provides an immune modulatorcomposition or a pharmaceutical composition comprising a whole cell of abacterium a rough strain of Mycobacterium obuense for use in enhancingthe immune system which may result in for example, enhancement of growthor an increase in the efficiency of feed utilisation in, for example,livestock.

In a further aspect, the present invention provides an immune modulatorcomposition or a pharmaceutical composition comprising a whole cell of abacterium a rough strain of Mycobacterium obuense for use in thetreatment or prevention of an immune system imbalance in children.

In a further aspect, the present invention provides an immune modulatorcomposition or a pharmaceutical composition comprising a whole cell of abacterium from a rough strain of Mycobacterium obuense for use in thetreatment or prevention of an adverse reaction to childhoodvaccines—such as whooping cough vaccinations and the current MMRvaccinations—and/or consequences thereof.

In another aspect, the present invention provides the use of a wholecell of a bacterium from a rough strain of Mycobacterium obuense in avaccine or a medicament, wherein said whole cell of said bacteriummodifies a cellular immune response.

In one aspect, the whole cell of a rough strain of Mycobacterium obuenseaccording to the present invention may downregulate a Th2 response.

In another aspect, the whole cell of a rough strain of Mycobacteriumobuense according to the present invention may upregulate a Th1response.

Suitably, the whole cell of a rough strain of Mycobacterium obuenseaccording to the present invention may downregulate a Th2 response andupregulate a Th1 response.

Alternatively, the whole cell of a rough strain of Mycobacterium obuenseaccording to the present invention may upregulate a Th1 response whilstnot affecting a Th2 response.

Alternatively, the whole cell of a rough strain of Mycobacterium obuenseaccording to the present invention may downregulate a Th2 response,whilst also downregulating a Th1 response.

Alternatively, the whole cell of a rough strain of Mycobacterium obuenseaccording to the present invention may upregulate a Th2 response, whilstalso upregulating a Th1 response.

In another aspect, the present invention provides a method for treatingor preventing a condition in a subject comprising administering aneffective amount of a pharmaceutical composition and/or immune modulatorcomposition to a subject wherein the said composition modulates acellular immune response.

Suitably the effective amount of the pharmaceutical composition and/orimmune modulator composition may be administered as a single dose.Alternatively, the effective amount of the pharmaceutical compositionand/or immune modulator composition may be administered in multiple(repeat) doses, for example two or more, three or more, four or more,five or more, ten or more, or twenty or more repeat doses.

In a further aspect, the present invention provides a method forimmunising a subject comprising administering a pharmaceuticalcomposition and/or immune modulator composition according to the presentinvention to a subject.

In a further aspect of the present invention, there is provided a methodfor protecting, including immunising, a subject comprising administeringa pharmaceutical composition and/or immune modulator compositionaccording to the present invention.

Preferably, a subject is protected, for example is immunised, againstone or more of: an infection (e.g. a bacterial, viral, for example aninfection caused by papilloma viruses, including equine sarcoid, genitalwarts and dysplasia of the uterine cervix that precedes carcinoma of thecervix, or parasitic infection, for example, malaria, trypanosomiasis,leishmaniasis, infection with Eimeria species in poultry andtoxoplasmosis) and/or the immunological abnormalities accompanying aninfection; an autoimmune disease (e.g. a vascular disorder, such asobliterative vascular disorder and the immunological aspects underlyingmyointimal hyperplasia and/or atheroma formation (otherwise known asarteriosclerosis), arthritis and graft rejection); stress (for example,major trauma stress, psychosocial stress and chronic stress); an allergy(e.g. asthma including allergic asthma, hayfever, allergic dermatitis(eczema), anaphylactic shock, allergies to plant contact or ingestion,stings—such as nettle and insect stings, and allergies to insectbites—such as midges, for instance Culicoides (which causes Sweet Itchin horses)); heaves; COPD; PMWS; PDNS; cancer (for example melanoma oradenocarcinoma or virally related cancers, such as cervical cancer); animmune system imbalance (e.g. an immune system imbalance in children andthe elderly); and post-operative stress and post-operative infection.

Preferably, a subject is immunised against one or more of malaria,trypanosomiasis, leishmaniasis, infection with Eimeria species inpoultry and toxoplasmosis.

Preferably, a subject is immunised against viral infections, for exampleagainst papilloma virus infections, including against equine sarcoid,genital warts or dysplasia of the uterine cervix that precedes carcinomaof the cervix.

Preferably, a subject is immunised against one or more of an autoimmunedisease, a vascular disorder, such as an obliterative vascular disorderand the immunological aspects underlying myointimal hyperplasia and/oratheroma formation (otherwise known as arteriosclerosis), arthritis andgraft rejection.

Preferably, a subject is protected against stress, such as, for example,one or more of major trauma stress, psychosocial stress and chronicstress and/or post-operative stress.

Preferably, a subject is protected (including immunised) against one ormore of asthma (including allergic asthma), hayfever, allergicdermatitis (eczema), anaphylactic shock, allergies to plant contact oringestion, stings—such as nettle and insect stings, and allergies toinsect bites—such as midges for instance Culicoides (which causes SweetItch in horses). More preferably, a subject is immunised against asthmaincluding for example allergic asthma, and allergies to insectbites—such as midges, for instance Culicoides(which causes Sweet Itch inhorses.

Preferably, a subject is protected (including immunised) against one ormore of COPD and/or heaves, particularly wherein the subject in a horse.

Preferably, a subject is protected (including immunised) against one ormore of PMWS and/or PDNS, particularly wherein the subject in a pig.

Preferably, a subject is protected (including immunised) against SIPH,particularly wherein the subject in a fish (such as koi), includingEIPH, particularly wherein the subject is a racing animal (such as ahuman, horse, camel or greyhound).

Preferably, a subject is protected (including immunised) againstpost-operative stress and infection.

Preferably, a subject is protected against the development and/or theprogression of a cancer, for example melanoma and/or adenocarcinomaand/or virally related cancers such as cervical cancers for example.

Preferably, a subject is protected (including immunised) against animmune system imbalance in the elderly. In particular, the compositionaccording to the present invention may be used to regulate the subject'simmune system.

Preferably, a subject is protected (including immunised) against animmune system imbalance in children. In particular, the compositionaccording to the present invention may be used to regulate the subject'simmune system, particularly the child's immune system.

Preferably, a subject is protected (including immunised) against anadverse reaction to childhood vaccines and/or consequences thereof. Inparticular, the immune system of the subject is regulated, particularlya child's immune system, before and/or during and/or afteradministration of the childhood vaccine.

The term “protected” as used herein means that the subject is lesssusceptible to the disease/disorder as compared with a subject nottreated or administered with the compositions according to the presentinvention and/or that the subject is more able to counter or overcomethe disease/disorder as compared with a subject not treated oradministered with the compositions according to the present invention.

In another aspect, the present invention provides administering aneffective amount of a pharmaceutical composition and/or an immunemodulator composition according to the present invention to a subject,wherein said composition is co-administered with an antigen or antigenicdeterminant.

When the composition is co-administered with an antigen or antigenicdeterminant in accordance with the present invention the antigen orantigenic determinant may suitably be an antigen or antigenicdeterminant from one or more of the following: BCG (bacillus of Calmetteand Guerin) vaccine, diphtheria toxoid vaccine,diphtheria/tetanus/pertussis (DTP or Triple) vaccine, pertussis vaccine,tetanus toxoid vaccine, measles vaccine, mumps vaccine, rubella vaccine,OPV (oral poliomyelitis vaccine), Mycobacterium vaccae, or part thereof(as taught in GB0025694.1) and a generic plasmodium antigen, for examplea malaria parasite antigen. Suitably two or more, or three or more, ofsuch antigens or antigenic determinants may be co-administered with apharmaceutical composition or an immune modulator composition accordingto the present invention.

Preferably, a medicament according to the present invention is used forthe treatment or prevention of one or more of: an infection (e.g. abacterial, viral, for example an infection caused by papilloma viruses,including equine sarcoid, genital warts and dysplasia of the uterinecervix that precedes carcinoma of the cervix, or parasitic infection,for example, malaria, trypanosomiasis, leishmaniasis, infection withEimeria species in poultry and toxoplasmosis) and/or the immunologicalabnormalities accompanying an infection; an autoimmune disease (e.g. avascular disorder, such as obliterative vascular disorder, arthritis andgraft rejection); stress (for example, major trauma stress, psychosocialstress and chronic stress; an allergy (e.g. asthma including allergicasthma, hayfever, allergic dermatitis (eczema), anaphylactic shock,allergies to plant contact or ingestion, stings—such as nettle andinsect stings, and allergies to insect bites—such as midges, forinstance Culicoides (which causes Sweet Itch in horses)); heaves; COPD;PMWS; PDNS; SIPH and cancer (for example melanoma or adenocarcinoma or avirally related cancer such as cervical cancers for example); an immunesystem imbalance (e.g. an immune system imbalance in children and theelderly); and post-operative stress and post-operative infection.

Preferably, a medicament according to the present invention is used forthe treatment or prevention of one or more of malaria, trypanosomiasis,leishmaniasis, infection with Eimeria species in poultry andtoxoplasmosis.

Preferably, a medicament according to the present invention is used forthe treatment or prevention of viral infections, for example papillomavirus infections, including equine sarcoid, genital warts or dysplasiaof the uterine cervix that precedes carcinoma of the cervix for example.

Preferably, a medicament according to the present invention is used forthe treatment or prevention of one or more of an autoimmune disease, anobliterative vascular disorder and the immunological aspects underlyingmyointimal hyperplasia and/or atheroma formation (otherwise known asarteriosclerosis), arthritis and graft rejection.

Preferably, a medicament according to the present invention is used forthe treatment or prevention of stress, such as, for example, majortrauma stress, psychosocial stress and chronic stress.

Preferably, a medicament according to the present invention is used forthe treatment or prevention of one or more of asthma (including allergicasthma), hayfever, allergic dermatitis (eczema), anaphylactic shock,allergies to plant contact or ingestion, stings—such as nettle andinsect stings, and allergies to insect bites—such as midges, forinstance Culicoides(which causes Sweet Itch in horses). More preferably,a medicament according to the present invention is used for thetreatment or prevention of asthma, including allergic asthma, andallergies to insect bites—such as midges, for instance Culicoides(whichcauses Sweet Itch in horses).

Preferably, a medicament according to the present invention is used forthe treatment or prevention of one or more of COPD and heaves.

Preferably, a medicament according to the present invention is used forthe treatment or prevention of one or more of PMWS and PDNS.

Preferably, a medicament according to the present invention is used forthe treatment or prevention of SIPH.

Preferably, a medicament according to the present invention is used forthe treatment or prevention of cancers (for example melanoma and/oradenocarcinoma and/or virally related cancers such as cervical cancersfor example).

Preferably, a medicament according to the present invention is used forthe treatment or prevention of an immune system imbalance in theelderly.

Preferably, a medicament according to the present invention is used forthe treatment or prevention of post-operative stress or infection.

Preferably, a medicament according to the present invention is used forthe treatment or prevention of an immune system imbalance in children.

Preferably, a medicament according to the present invention is used forthe treatment or prevention of an adverse reaction to childhood vaccinesand/or consequences thereof.

In a further aspect of the present invention, a pharmaceuticalcomposition or an immune modulator composition according to the presentinvention may comprise bacteria from a rough strain of Mycobacteriumobuense. Suitably, the composition may comprise two or more, or three ormore, bacteria from a rough strain of Mycobacterium obuense.

Suitably, the bacterium for use in accordance with the present inventionmay be used in conjunction with other immune enhancing bacteria such asbacteria from any of the following genera Rhodococcus, Gordonia,Nocardia, Dietzia, Tsukamurella and Nocardioides including any speciesfrom any of these genera such as, Gordonia bronchialis, G. amarae, G.sputti, G. terrae, Nocardia asteroides, Dietzia maris, Tsukamurellapaurometabola, Rhodococcus ruber, Rhodococcus rhodnii, R. coprophilus,Nocardioides albus and Tsukamurella inchonensis for example.

Suitably, the species used from each particular genus are ones which canbe grown on medium, which is a low, preferably non-, antigenic medium.By way of example only, a suitable non-antigenic medium is Sauton'smedium.

More preferably, bacteria to be used in conjunction with the bacteriafor use in accordance with the present invention are from the genusRhodococcus. including Rhodococcus ruber (previously known as Nocardiarubra), Rhodococcus rhodocrous, Rhodococcus rhodnii, Rhodococcuscoprophilus, Rhodococcus opacus, Rhodococcus erythopolis.

More preferably, a bacterium to be used in conjunction with the bacteriafor use in accordance with the present invention is Rhodococcus ruber.

By way of example only, one or more of the organisms Gordoniabronchialis, Rhodococcus ruber, Rhodococcus rhodocrous, Rhodococcusrhodnii, Dietzia maris and Gordonia terrae in conjunction with thebacteria for use in the present invention may be effective in thetreatment and/or prevention of parasitic infections.

By way of example only, one or more of the organisms Tsukamurellainchonensis, Gordonia amarae and Nocardia asteroids in conjunction withthe bacteria for use in the present invention may be particularlyeffective in the treatment and/or prevention of allergies, such asallergies to insect bites—such as midges for example, and/or thetreatment and/or prevention of cancers, including skin neoplasms such asEquine sarcoid.

By way of example only, Rhodococcus coprophilus in conjunction with thebacteria for use in the present invention may be particularly effectivein the modulation of infections, in particular parasitic infections,and/or enhancing growth in livestock.

In one embodiment of the present invention whole cells of a bacteriumfrom may be used in conjunction with Rhodococcus coprophilus in theprevention or treatment of PMWS and/or PDNS.

In one embodiment of the present invention whole cells of a bacteriumfrom a rough strain of Mycobacterium obuense may be used in conjunctionwith Tsukamurella inchonensis in the prevention or treatment of SIPH.

Preferably, the bacterium according to the present invention is killedprior to use.

Preferably, the bacterium according to the present invention is killedby heat-treatment thereof, for example, heat-treatment in an autoclaveat 121° C. for 15 minutes. Other suitable treatments for killing thebacterium may include ultraviolet or ionising radiation or treatmentwith chemicals such as phenol, alcohol or formalin. Suitably, theionising radiation may be carried out by exposure to 2.5 Mrads from aCo₆₀ source.

Preferably, the bacterium according to the present invention is purifiedand/or isolated.

Preferably, the bacterium according to the present invention issuspended in water or buffered saline, suitably borate buffered at pH 8.

The term “subject”, as used herein, means an animal. Preferably, thesubject is a mammal, bird, fish or crustacean including for examplelivestock and humans. In some aspects of the present invention, thesubject may suitably be a human.

In an aspect of the present invention, for example in the treatment ofPMWS and/or PDNS, the subject may be a pig.

In another aspect of the present invention, for example in the treatmentof COPD, heaves and/or Culicoides, the subject may be a horse.

In another aspect of the present invention, for example in the treatmentof SIPH, the subject may be a fish (such as koi) or a racing animal(such as a human, horse, camel or greyhound). Preferably the subject isa racing animal.

The term “immune modulator” as used herein includes a vaccine.

In some embodiments of the present invention, the term “rough strain”refers to a stable rough strain. By “stable” it is meant that a roughstrain retains it rough colonial morphology for greater than or equal to20 successional cultures.

The term “colonial morphology” or “colony morphology” as used hereinmeans the cultural characteristics of a bacterium on an agar plate.

The term “rough” as used herein means other than smooth. The term“rough” as used herein may include characteristics such as an irregularcolony morphology, and may include for instance undulate and/or lobatemorphology.

In another embodiment of the present invention, the term “rough” as usedherein means that the strain is unable and/or substantially unable toproduce O-polysaccharide.

Producing Rough Strains

Some aspects of the present invention relate to methods of producing arough stain of bacteria by exposing bacteria to a sulfone and/or asulfonamide or the use of a sulfone and/or a sulfonamide to producerough strains.

Such methods and uses are predicated on the surprising finding thatexposure of a bacterium (such as a smooth variant of Mycobacterium) asulfone and/or a sulfonamide results in the production of roughvariants.

Preferably, a bacterium for use in a method of the present invention isfrom a genus of aerobic organisms in the order of Actinomycetales.Preferably, said bacterium is from the genus Mycobacterium. Suitably,said bacterium is from Mycobacterium obuense.

Suitably, a bacterium for use in the method of the present invention maybe grown on any suitable medium, such as Middlebrook 7H11 medium.

Preferably a bacterium for use in the present invention is exposed to aconcentration of a sulfone and/or a sulfonamide at a concentration ofgreater than or equal to a sulfone and/or a sulfonamide per 1 ml medium.Preferably at a concentration of greater than or equal to 7.5 μg/ml.Preferably at a concentration of greater than or equal to 10 g/ml.Preferably at a concentration of less than 30 μg/ml. Preferably at aconcentration or equal to or less than 20 μg/ml. Preferably at about 12μg/ml.

Suitably, a sulfone and/or a sulfonamide may be admixed with a suitablemedium and a bacterium may be grown thereon.

Rough colonies of a bacterium produced by a method of the presentinvention may further be isolated.

Suitably, rough colonies of a bacterium produced by a method of thepresent invention may be stable.

In some embodiments of the present invention the term “rough strain”refers to a strain of bacteria that is able to retain a rough morphologyon at least 3 repeated cultures in the absence of sulfone and/orsulfonamide thereof.

Sulfone

The term sulfone refers to a compound of the formula (I):

whereinR₁ is a hydrocarbyl group,R₂ is a hydrocarbyl group.

Preferably, R₁ is selected from an alkyl, a substituted alkyl, an aryl,a substituted aryl, a heteroaryl and a substituted heteroaryl group.

Preferably, R₁ is selected from an aryl, a substituted aryl, aheteroaryl and a substituted heteroaryl group.

Preferably, R₁ is a substituted phenyl group.

Preferably, R₁ is a 4-amino-phenyl group.

Preferably, R₂ is selected from an alkyl, a substituted alkyl, an aryl,a substituted aryl, a heteroaryl and a substituted heteroaryl group.

Preferably, R₂ is selected from an aryl, a substituted aryl, aheteroaryl and a substituted heteroaryl group.

Preferably, R₂ is a substituted phenyl group.

Preferably, R₂ is a 4-amino-phenyl group.

R₁ and R₂ may be the same or different.

In one preferred embodiment, the sulfone is 4,4′-diaminodiphenyl sulfoneand/or an analogue thereof.

4,4′-Diaminodiphenyl sulfone is commonly marketed by Sigma under thetrade mark Dapsone™.

Synonyms for 4,4′-Diaminodiphenyl sulfone include N,N′-Diphenylsulfondiamide; 4,4′-Sulfonyldianiline; Di(p-aminophenyl) sulfone;Novophone; Diaphenylsulfon; Dumitone; Diphone; 4-Aminophenyl sulfone;Bis(p-aminophenyl) sulfone; WR 448; F 1358; Croysulfone; Diphenasone;Metabolite C; Dapsone(USAN); 1,1′-Sulfonylbis[4-aminobenzene];Diaminodiphenyl sulfone; Di(4-aminophenyl) sulfone; p,p′-Sulfonyldianiline; Bis(4-aminophenyl) sulfone;4,4′-Sulfonylbisbenzamine; Eporal; DADPS; diaphenylsulfone; dapsone;Avlosulfon; Benzenamine, 4,4′-sulfonylbis-; Aniline, 4,4′-sulfonyldi-;Udolac; DDS, pharmaceutical; DSS; DDS; p,p′-Diaminodiphenyl sulfone;Sulfadione; Disulone; Avlosulfone; p-Aminophenyl sulfone; Dapson;Sulfona; NSC 6091D; and Diaminodiphenylsulfone.

The term “Dapsone™” as used herein refers to N,N′-Diphenyl sulfondiamideand relates to any compound that has the following formula:

Sulfonamide

The term sulfonamide refers to a compound of the formula (II):

whereinR₁ is a hydrocarbyl group,R₃ is a hydrogen or a hydrocarbyl group,R₄ is a hydrogen or a hydrocarbyl group.

Preferably, R₁ is selected from an alkyl, a substituted alkyl, an aryl,a substituted aryl, a heteroaryl and a substituted heteroaryl group.

Preferably, R₁ is selected from an aryl, a substituted aryl, aheteroaryl and a substituted heteroaryl group.

Preferably, R₁ is a substituted phenyl group.

Preferably, R₁ is a 4-amino-phenyl group.

Preferably, R₃ is selected from hydrogen, an alkyl, a substituted alkyl,an aryl, a substituted aryl, a heteroaryl and a substituted heteroarylgroup.

Preferably, R₃ is selected from an aryl, a substituted aryl, aheteroaryl and a substituted heteroaryl group.

Preferably, R₃ is a substituted phenyl group.

Preferably, R₃ is a 4-amino-phenyl group.

Preferably, R₄ is selected from hydrogen, an alkyl, a substituted alkyl,an aryl, a substituted aryl, a heteroaryl and a substituted heteroarylgroup.

In one embodiment, at least one of R₃ and R₄ is hydrogen.

In another embodiment, at least one of R₃ and R₄ is an alkyl or asubstituted alkyl group.

DEFINITIONS

The term “hydrocarbyl group” as used herein means a group comprising atleast C and H and may optionally comprise one or more other suitablesubstituents. Examples of such substituents may include halo-, alkoxy-,nitro-, an amino group, a hydrocarbon group, an N-acyl group, a cyclicgroup etc. In addition to the possibility of the substituents being acyclic group, a combination of substituents may form a cyclic group. Ifthe hydrocarbyl group comprises more than one C then those carbons neednot necessarily be linked to each other. For example, at least two ofthe carbons may be linked via a suitable element or group. Thus, thehydrocarbyl group may contain hetero atoms. Suitable hetero atoms willbe apparent to those skilled in the art and include, for instance,sulphur, nitrogen and oxygen.

In one preferred embodiment of the present invention, the hydrocarbylgroup is selected from an alkyl, a substituted alkyl, an aryl, asubstituted aryl, a heteroaryl and a substituted heteroaryl group.

The term “alkyl” refers to straight chain, branched chain or cyclichydrocarbon groups having 1 to 12 carbon atoms, preferably 1 to 8 carbonatoms, such as methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl,t-butyl, pentyl, hexyl, heptyl, octyl, etc. Lower alkyl groups, that is,alkyl groups of 1 to 6 carbon atoms, are most preferred.

The term “substituted alkyl” refers to alkyl groups substituted withone, two or three groups selected from halogen, nitro, cyano, hydroxy,haloalkyl, haloalkoxy, —OR_(x), —NR_(x)R_(y), —SR_(x), —C(═O)R_(x),—OC(═O)R_(x), —S(═O)R_(x), —SO₂R_(x), —SO₃R_(x), —CO₂R_(x),—NR_(x)C(═O)R_(y), —NR_(x)CO₂R_(y), —NR_(x)SO₂R_(y), —SO₂NR_(x)R_(y), or—C(═O)NR_(x)R_(y), wherein the or each R_(x) is independently selectedfrom hydrogen, C₁₋₆alkyl, C₂₋₆alkenyl, aryl, heteroaryl and benzyl;wherein the or each R_(y) is independently selected from hydrogen,C₁₋₆alkyl, C₂₋₆alkenyl, aryl, heteroaryl and benzyl.

The term “aryl” refers to phenyl, 1-naphthyl and 2-naphthyl, with phenylbeing preferred, as well as such rings having fused thereto acycloalkyl, cycloalkenyl, heterocyclo, or heteroaryl ring.

The term “heteroaryl” refers to aromatic 5 or 6 membered monocyclicgroups, 9 or 10 membered bicyclic groups, and 11 to 14 memberedtricyclic groups which have at least one heteroatom (O, S or N) in atleast one of the rings. Each ring of the heteroaryl group containing aheteroatom can contain one or two oxygen or sulfur atoms and/or from oneto four nitrogen atoms, provided that the total number of heteroatoms ineach ring is four or less and each ring has at least one carbon atom.The fused rings completing the bicyclic and tricyclic groups may containonly carbon atoms and may be saturated, partially saturated, orunsaturated. The nitrogen and sulfur atoms may optionally be oxidizedand the nitrogen atoms may optionally be quaternized. Heteroaryl groupswhich are bicyclic or tricyclic must include at least one fully aromaticring but the other fused ring or rings may be aromatic or non-aromatic.The heteroaryl group may be attached at any available nitrogen or carbonatom of any ring. Examples of heteroaryl rings include, withoutlimitation: furan, thiophene, pyrrole, oxazole, thiazole, imidazole,isoxazole, pyrazole, isothiazole and pyridine.

The term “heteroatoms” shall include oxygen, sulfur and nitrogen.

The terms “substituted aryl” and “substituted heteroaryl” includes suchrings having one, two or three suitable substituents.

The term “analogues of 4,4′-diaminodiphenyl sulfone” means a compoundwith the basic structure of 4,4′-diaminodiphenyl sulfone wherein fromone to six of the hydrogen atoms and/or amino groups attached to thephenyl rings have been substituted with a suitable substituent, and nomore that three suitable substituents appear on either of the phenylrings. Suitable substituents for analogues of 4,4′-diaminodiphenylsulfone may include substituting an amino group with a hydrogen.

Preferably the or each suitable substituent is selected from C₁₋₆alkyl,C₂₋₆alkenyl, phenyl, benzyl, halogen, nitro, cyano, hydroxy, haloalkyl,haloalkoxy, R_(x), —NR_(x)R_(y), —SR_(x), —C(═O)R_(x), —OC(═O)R_(x),—S(═O)R_(x), —SO₂R_(x), —SO₃R_(x), —CO₂R_(x), —NR_(x)C(═O)R_(y),—NR_(x)CO₂R_(y), —NR_(x)SO₂R_(y), SO₂NR_(x)R_(y), or —C(═O)NR_(x)R_(y),wherein the or each R_(x) is independently selected from hydrogen,C₁₋₆alkyl, C₂₋₆alkenyl, aryl, heteroaryl and benzyl; wherein the or eachR_(y) is independently selected from hydrogen, C₁₋₆alkyl, C₂₋₆alkenyl,aryl, heteroaryl and benzyl.

Preferably the or each suitable substituent is —NR_(x)R_(y), whereinR_(x) and R_(y) are as defined above.

Preferably, the or each suitable substituent is —NH₂.

When a subscript is used in conjunction with a group such as C₁₋₄alkyl,the subscript refers to the number of carbon atoms that the group willcontain, in addition to heteroatoms. Thus, the term hydroxyC₁₋₄alkyl orC₁₋₄hydroxyalkyl refers to an alkyl group of one to four carbon atomshaving an OH substituent on one of the carbon atoms.

The term “alkenyl” refers to straight chain, branched chain or cyclichydrocarbon groups having at least one double bond. Alkenyl groups of 2to 6 carbon atoms and having one double bond are most preferred.

The term “halo” or “halogen” refers to chloro, bromo, fluoro and iodo.

The term “haloalkyl” means an alkyl having one or more halosubstituents. Thus, it includes, for example, trifluoromethyl. The term“perfluoromethyl” means a methyl group having two or three fluorosubstituents.

The term “haloalkoxy” means an alkoxy group having one or more halosubstituents. For example, “haloalkoxy” includes —OCF₃.

Therapeutic Uses

The immune modulators of the present invention may be used in therapy.In particular such compounds may be used to modulate T lymphocyteresponses in vivo and/or other cells involved in an immune response invivo.

Immune modulator/pharmaceutical compositions capable of modulating, inparticular blocking, T cell proliferation and/or differentiation and/oractivity may be used against any disorder which is susceptible toprevention or treatment by the modulation of an adaptive immuneresponse, i.e. a cellular immune response.

Suitably, the compositions according to the present invention are usedto modulate a cellular immune response to treat or prevent one or moreof: an infectious disease (such as a bacterial infection e.g.methycillin-resistant Staphylococcus aureus, tuberculosis, includingmultidrug resistant tuberculosis and leprosy; chronic viral infections,for example hepatitis, HIV and infections caused by papilloma viruses,including equine sarcoid, genital warts and dysplasia of the uterinecervix that precedes carcinoma of the cervix; latent viral infections,such as shingles (Herpes zoster) or cold sores (Herpes simplex) forexample; or parasitic infections, for example malaria, trypanosomiasis,leishmaniasis, infection with Eimeria species in poultry andtoxoplasmosis); an allergy (such as allergic dermatitis or allergicasthma); an autoimmune disease (e.g. a vascular disorder, such asobliterative vascular disorder, arthritis and graft rejection); stress(such as major trauma stress, psychosocial stress and chronic stress);PMWS, PDNS, SIPH and cancer (for example, the composition may beadministered regularly throughout adult life to counter the effects oftobacco).

A more extensive list of disorders is given in WO-A-98/09985. For easeof reference, part of that list is now provided: inflammation associatedwith hypersensitivity, allergic reactions, asthma, inflammationassociated with aphthus ulceration, ulcerative colitis, hepaticfibrosis, liver cirrhosis or other hepatic diseases, dermatitis, inparticular atopic dermatitis e.g. eczema, periodontal diseases or otherdental diseases, to treat or ameliorate monocyte or leukocyteproliferative diseases, e.g. leukaemia, other cancers.

Infectious Diseases

Compositions capable of modulating, in particular stimulating (i.e.inducing or enhancing) T cell proliferation and/or differentiation or ofpreventing the induction of or reversing T cell anergy may be usedgenerally to boost or induce T cell immune responses. Virtually alladaptive immune responses require the activation of T cells and theirdifferentiation into cytokine-producing cells. Thus, these compositionsmay be used generally to prevent and/or treat infectious diseases—suchas viral or bacterial. Suitably, these compositions may be used toprevent and treat parasitic infections, e.g. malaria, leishmaniasis,toxoplasmosis and trypanosomiasis). Suitably, these compositions may beused to prevent or treat viral infections, for example infections causedby papilloma viruses, including equine sarcoid, genital warts anddysplasia of the uterine cervix that precedes carcinoma of the cervix.

In one aspect of the present invention the infection is preferablytrypanosomiasis.

In another aspect of the present invention the infection is preferablyone caused by papilloma viruses, particularly bovine papilloma viruses 1and 2. In a further aspect of the present invention the infection ispreferably one or more of equine sarcoid, genital warts and dysplasia ofthe uterine cervix that precedes carcinoma of the cervix.

Equine Sarcoid

The compositions of the present invention may also be used to preventand treat equine sarcoid.

Equine sarcoid is the commonest skin neoplasm of horses and isassociated with infection with bovine papilloma viruses 1 and 2(Chambers et al J. Gen. Virol. 2003: 84: 1055-1062). This condition iscurrently without an effective treatment, although surgery, non-specificimmune treatment and cytotoxic drugs may all have some effect.

The compositions of the present invention are preferably administered inthe same lymph node drainage area as a lesion or neoplasm.

PMWS and PDNS

Post-weaning multisystemic wasting syndrome (PMWS) affects pigletspost-weaning from 4 to 16 weeks of age (15-50 kg). Typically PMWSaffects piglets one to two weeks after weaning and is very differentfrom the wasting/poor weaner who fails to eat or drink adequately afterweaning. PMWS piglets are weaners which have started to grow and thencollapse quickly and often have an extremely poor response toantibiotics.

Porcine dermatitis and nephropathy syndrome (PDNS) affects pigs from 8to 18 weeks of age and the most obvious signs are red-purple blotches onthe skin, which become brown and crusted after a few days. Pigs arelethargic and may have swollen legs resulting from their nephropathy.This syndrome, also, responds poorly to antibiotics.

The causal agents of both PMWS and PDNS are at present unknown. The mostlikely suspect in both syndromes is a pig circovirus “type II” which isantigenically distinct from widely distributed normal non-pathogenic pigcircovirus “type I”. Circovirus II (PCV II) has been identified on UKfarms serologically. PDNS, which is thought to be an immune complexmediated disease, may also involve bacteria in its aetiology, though thepart that they play is not clear

Suitably the compositions of the present invention may be used in themanufacture of a medicament for the treatment or prevention of PMWSand/or PDNS.

SIPH

The compositions of the present invention may also be used to preventand treat SIPH. invention may also be used to treat high-altitudepulmonary edema (HAPE).

Stress-induced pulmonary haemorrhage (SIPH) is a condition that causesbleeding from blood vessels within the lung when the animal is stressed.The term “SIPH” as used herein may encompass the condition HAPE (highaltitude pulmonary edema), which is a condition caused by stress-failureof the pulmonary capillaries allowing fluid uptake into the lung (alsoknown as wet-lung). In addition, the term “SIPH” as used herein may alsoencompasses exercise-induced pulmonary haemorrhage (EIPH), which is acondition that causes bleeding from blood vessels within the lung duringstrenuous exercise.

HAPE is a potentially fatal condition that typically occurs 2 to 4 daysafter ascent to altitudes above 3000m. With usual ascent rates, theincidence is about 1% to 2%, but as many as 10% of people ascendingrapidly to 4500 m may develop the condition. HAPE may be preceded byacute mountain sickness, but this is not always the case. Thepredominant symptom is dyspnea with reduced exercise tolerance. There isoften a dry cough at first, but this may progress to a cough thatproduces frothy, blood stained sputum. Tachypnea and tachycardia arecommon on examination.

EIPH is known to affect mammals, particularly racing mammals, such ashorses, greyhounds, camels and humans. EIPH is known to affect mammals,particularly racing mammals, after intense exercise.

EIPH is most widely described in thoroughbred horses, where it isthought to cause a loss of performance, but has also been observed instandardbred racing (trotting or pacing), polo, show jumping, crosscountry and barrel racing horses. EIPH is a common condition it isbelieved afflicting up to 85% of equine athletes.

The symptoms of EIPH vary from minor bleeding detected by observing redblood cells in broncho-alveolar fluid obtained by fibroscopy, to bloodappearing in mucus froth around the nostrils at the end of a race.Although in the most severe cases, EIPH manifests as bleeding from thenostrils (epistaxis) but many horses do not show any signs. The use ofendoscopy has shown that 40-75% of thoroughbred horses do have blood intheir trachea after racing. Diagnosis can also be achieved by trachealwashing and by bronchioalveolar lavage (BAL).

Allergy

The compositions of the present invention may also be used to preventand treat allergies (e.g. asthma including allergic asthma, hayfever,allergic dermatitis (eczema), anaphylactic shock, allergies to plantcontact or ingestion, stings—such as nettle and insect stings, andallergies to insect bites—such as midges for instance Culicoides(whichcauses Sweet Itch in horses).

Sweet Itch is one of the commonest skin diseases seen in horses,particularly in wild horses and/or ponies. About 3% of horses in theU.K. are affected to some degree. Most horses show signs between 1 and 4years of age and the condition generally worsens during summer. Certainbreeds are particularly prone to the disease. Shires, Hackneys and Welshand Icelandic ponies have all been suggested as susceptible breeds.Sweet Itch is caused by hypersensitivity to the bites of the tiny flyCulicoides. In the UK, the fly is present from April to October butpeaks in numbers in May to September. The flies feed on the horse atspecific sites usually around the tail head and under the mane. Thereare 20 species of Culicoides present in the UK and some feed underneaththe horses' abdomen.

By way of example only, the compositions of the present invention mayalso be used to prevent and/or treat anaphylactic shock by administeringa subject, which subject has a predisposition to suffer fromanaphylactic shock (for instance a subject who is known to have anallergy, such as an allergy to peanuts for instance) with a compositionaccording to the present invention, thus to reduce the subject'spredisposition to anaphylactic shock should they (accidentally) comeinto contact with the antigen, e.g. peanuts for instance, towards whichthey may react adversely.

Heaves/COPD

The compositions of the present invention may also be used to preventand treat heaves and/or COPD (Chronic Obstructive Pulmonary Disease).

Heaves is an equine lung disease with similarities to human asthma andCOPD. The clinical signs in the horse are initiated by an allergicresponse to the particles in hay dust in lungs already damaged with adegree of fibrosis. It is most often seen in older horses (greater thansix years old) that are stabled during the winter months. Hay containsmicroorganisms—such as bacteria and fungi as well as tiny particles offeed grains, plants, faeces, dander, and pollen. These tiny particlesbecome aerosolised in hay dust and elicit an allergic response andfibrosis when they are inhaled by horses with heaves. The primarymicroorganisms believed to be involved in the etiology of heaves areAspergillus fumigatus, Thermoactinomyces vulgaris and Faeniarectivirgula. Both reduction of the bronchospasm of asthma and thefibrosis of COPD are within the scope of the patent.

Autoimmune Diseases

The compositions of the present invention may be used to treat and/orprevent an autoimmune disease mechanistically related to poor T cellregulation and/or T cell dysregulation. Examples of autoimmune diseasesinclude one or more of the following: unwanted immune reactions andinflammation including arthritis, including rheumatoid arthritis,psoriasis, psoriatic arthropathy, vascular disorders, in particular avascular disorder in which there is inflammation of the intima of theblood vessel, examples of vascular disorders are atheroma formation(otherwise known as arteriosclerosis), anterior uveitis and myointimalhyperplasia following angioplasty; thyroiditis, atherosclerotic heartdisease, reperfusion injury, cardiac conduction disturbances, myocardialinfarction, habitual abortion, retinitis pigmentosa, immune andinflammatory components of degenerative fundus disease, inflammationassociated with autoimmune diseases or conditions or disorders where,both in the central nervous system (CNS) or in any other organ, immuneand/or inflammation suppression would be beneficial, Parkinson'sdisease, complication and/or side effects from treatment of Parkinson'sdisease, Guillaim-Barre syndrome, myasthenia gravis, graft rejection incases of transplantation of natural or artificial cells, tissue andorgans such as cornea, bone marrow, organs, lenses, pacemakers, naturalor artificial skin tissue.

In more detail: Organ-specific autoimmune diseases include multiplesclerosis and inflammatory bowel diseases (Crohn's disease, ulcerativecolitis) for example.

Systemic autoimmune diseases include: rheumatoid arthritis.

Vascular disorders include vascular disorders in which there isinflammation of the intima of the blood vessels.

Suitably, the vascular disorders according to the present invention mayinclude any vascular disease or disorder which comprises an autoimmuneelement, for example one which is caused by an autoimmune response.

Suitably, vascular disorders according to the present invention mayinclude one or more of Raynaud's disease and phenomenon, anterioruveitis, obliterative vascular disorder, atheroma formation (otherwiseknown as arteriosclerosis), arteritis, myointimal hyperplasia (naturalor following angioplasty), inflammatory and autoimmune thickening of theintima and/or muscular layer of blood vessels, inflammatory blood vessellesions, atherosclerotic heart disease, reperfusion injury, cardiacconduction disturbances, myocardial infarction.

Suitably, the graft rejection according to the present invention may bechronic graft rejection, particularly in the absence of animmunosuppressant. Thus, the composition according to the presentinvention may be used as a replacement for the conventionalimmunosuppressant administered prior to, during and/or aftertransplantation. The compositions according to the present invention maybe used when transplanting natural or artificial cells, tissues andorgans, such as one or more of the following: corneas, bone marrow,organs (e.g. kidney, liver), lenses, pacemakers, natural or artificialskin tissue, islet cells.

Preferably, the compositions of the present invention may be used totreat the following autoimmune diseases: a vascular disorder, arthritis,graft rejection and the immunological aspects underlying myointimalhyperplasis and atheroma formation.

Stress

Stress is often presented as a symptom of modern living, the highpressure executive lifestyle, the consequences of which are widelyperceived as leading to major pathological conditions such as gastriculcers, hypertension, heart disease and strokes. Other major stressfulevents in life such as divorce, bereavement and moving house are seen ashigh risk factors for heart disease.

These are not misconceptions, the farming industry is well aware of theeconomic losses resulting from subjecting livestock to major stressessuch as overcrowding, confinement and transportation leading to anincreased susceptibility to infection and the precipitation ofunderlying pathology. Research by doctors and scientists is producing anincreasing volume of published work showing definable stresses such asconfinement can result in significant changes in endocrine (hormone)activity which subsequently can affect the body's immune functions. Thiscan be noticeably demonstrated in major trauma stress (includingsurgical stress) in which the cell mediated immune response isdramatically paralysed Faist (1996).

Elenkov I J (1999) report recent evidence indicating thatglucocorticoids and catecholamines, the end products of the stresssystem, and histamine, a product of activated mast cells, mightselectively suppress cellular immunity, and favour humoral immuneresponses. This is mediated by a differential effect of stress hormonesand histamine, on Th1/Th2 patterns and type 1/type 2-cytokineproduction. Thus, systemically, stress might induce a Th2 shift, while,locally, under certain conditions, it might induce pro-inflammatoryactivities through neural activation of the peripheralcorticotrophin-releasing factor-mast cell-histamine axis.

Paik (2000) and Kay (2001) in independent studies of academic stress,examined the immunological profiles of students during non exam and examperiods. They report a significant reduction in IL-2 and interferongamma production and an increase in IL-6.

This indicates that the body's immune system responds to stressfulepisodes by a down regulation of Th-I cytokines and a selectiveup-regulation of the Th2 cytokines.

Iwakabe (1998), using a mouse model of restraint stress reports theskewing of the immune response towards Th2 dominant immunity.

This stress hormone induced switch towards Th2 immune imbalance is alsoreported in non major, chronic stress situations such as psychosocialstress amongst workers overwintering at the Australian NationalAntarctic Research Expedition stations, (Mehta (2000)). They also reportan associated increase in latent virus reactivations.

Similar stress hormone and immunological changes are reported fromchronic stress in care givers of dementia patients (Bauer (2000)) and inastronauts during the Euromir 95 mission (Norbiato (1998)). Ofparticular concern was the astronauts increased susceptibility toinfection.

The body is designed to recover from stress and in acute stress clearlydoes as the risk of infection recedes with the patient's recovery fromthe major trauma.

Chronic stress however appears to maintain the Th2 dominated immuneimbalance. This is a very serious consequence as all of the quotedauthors allude to stress through the above mechanisms, possiblyinfluencing the onset and/or course of infectious, inflammatory,allergic and neoplastic diseases.

This consequence is further supported by Lawrence (2000).

An immunoregulator, preferably, an orally administered immunoregulator,according to the present invention, which stimulates the Th1 responseand down regulates Th2 may restore the healthy balance of the immunesystem and thus reduce the increased risk of serious illness associatedwith chronic stress.

Preferably, the composition according to the present invention is usedto treat and/or prevent stress, in particular major trauma stress,psychosocial stress and chronic stress.

Preferably, the composition according to the present invention is usedto treat and/or prevent stress in animals, suitably in humans and/orlivestock.

Immune System Imbalance

An immune system imbalance—such as an upregulation, downregulation orinappropriately regulated cellular immune response—may occur at any timein the life of a subject. Suitably, the compositions may be used tomodulate an immune system imbalance. That is to say, the compositionsaccording to the present invention may be used to treat and/or preventan immune system imbalance.

(a) In Children

Suitably, the immune modulator composition or a pharmaceuticalcomposition may be used to modulate an immune system imbalance, inchildren, including babies, infants and juveniles. An immune systemimbalance—such as an upregulation, downregulation or inappropriatelyregulated cellular immune response—may occur in children followingvaccination, for example following childhood vaccinations. Such animmune system imbalance may result in conditions such as the onset ofallergies, i.e. allergic dermatitis and allergic asthma.

With the aim of protecting children from infections, repeated injectionsagainst Diphtheria, Tetanus, Pertussis, Polio, Measles, Mumps and theRubella are given. All of these are judged necessary and pressure isexerted by Health authorities to ensure that children are presented forvaccination at the appropriate time. However, most vaccinations given inearly life contain an alum adjuvant, which has important immunologicalconsequences. Alum is a potent stimulus to the Th2 pattern of responseand the consequential immune dysregulation causes the child to becomevulnerable to the development of allergies and possibly cancer forexample.

It is possible to re-educate the immune system to a proper recognition,regulation and response both to self and to the rest of the world.

Suitably, the immune modulator composition or a pharmaceuticalcomposition may also be used for the treatment or prevention of anadverse reaction to childhood vaccines—such as whooping coughvaccinations and the current MMR vaccinations—and/or consequencesthereof.

(b) Immune System Imbalance in the Elderly

An immune system imbalance—such as an upregulation, downregulation orinappropriately regulated cellular immune response, in particulardownregulation, for example a deterioration of immune function—may occurin older people, generally in excess of 60 years. In elderly people, adownregulation in the cellular immune response is generally referred toas immunosenescence. Typically, the deterioration of immune function maylead to increased susceptibility to infectious diseases and neoplasiafor example. The number of old people as a proportion of the populationis dramatically increasing and geriatric medicine is becoming animportant aspect of clinical practice. It is not surprising thereforethat research has focused on the mechanisms of immunosenescence and thelinks between the health of the immune system and longevity. Goronzy(2001), examined the varying efficacy of influenza vaccination in theelderly. In this study, only 17% of subjects showed a rise in titre toall 3 haemaglutinins (successful vaccination) 1 month followingvaccination and that 46% showed no demonstrable response at all. It wasproposed that responsiveness to influenza vaccination is a usefulbiological marker of immunosenescence. A number of researchers havestudied various aspects of the immune function in the elderly. Forexample, Lio (2000) studied cytokine responses, Solana (2000) studied NKand NKt cells, and Ginaldi (1999) suggested that a Th1 to Th2 cytokineproduction shift and an increased production of proinflammatorycytokines could explain many aspects of age-associated pathologicalevents, such as atherosclerosis and osteoporosis. Accordingly, anon-pathological stimulation of the immune system which drives thecytokine response away from the proinflammatory Th2 towards Th1 isrequired. Preferably, such an immune modulator reduces the mortalityfrom acute infection, counters the onset and reduces the morbidity ofage related autoimmune disease and possibly reduces the rate ofneoplastic disease, all of which are associated with immunosenescence.

Typically, an immune modulator composition or a pharmaceuticalcomposition according to this aspect of the present invention may be animmune enhancer.

The potential role of probiotic commensal gut or dairy bacteria havebeen investigated in this area. For example, milk products supplementedwith 5×10⁹ or 5×10¹⁰ Bifidobacterium lactis or Lactobacillus rhamnosusper dose taken daily for 3 weeks has been reported to increase thenumbers of peripheral blood NK, CD4 and CD25 cells and generally boostsystemic cellular immunity in the elderly (Gill, (2001)).

Currently a number of products containing high numbers of Lactobacilliand other intestinal commensal flora are being actively promoted as“lifestyle enhancers”. A review by Sanders (2001), on the claimedprobiotic effects of Lactobacillus acidophilus, available as a drugsince 1950, suggests its effects require further validation andclarification of the mechanism of action. Whilst there is a beneficialeffect from replacing the bowel flora after diarrhoeal disease andcombating candidiasis following antibiotic therapy, immune stimulationappears unreliable and short lived. However, this work clearlyidentifies a role for a potent orally administered immune modulator,preferably killed so as to avoid the difficulties of maintaining liveproducts.

Oral vaccination is a long established successful mechanism for inducinglocal protective immunity against oral/faecal pathogens—such as polio.However, orally administered vaccines have also been shown to evokesystemic protective immune responses both cell mediated and humoral.Sharpe (2002), used an orally administered adenovirus constructcontaining measles virus antigen to induce systemic antibody and spleniclymphocyte responses to the measles antigen. Manube (2002) has developeda model to show that orally administered attenuated Mycobacteriummicroti provides a higher level of protection to an aerosol challengewith tuberculosis than traditional subcutaneously administered BCG.

Kim (2001) showed that feeding with Japanese cedar pollen produced oraltolerance to specific allergy induced by subsequent injection of pollenin oil. This was associated with decreased specific immunoglobin levelsand a significant reduction in interleukin-4 production i.e. the TH2response was down-regulated.

Therefore, a systemic immune response may be both stimulated andmodulated by administration—such as oral administration—of a suitableimmune modulator.

Suitably, in one aspect of the present invention it is envisaged that awhole cell of the bacterium according to the present invention, may beincluded in food preparations and/or may be supplied as a type of“remedy”, preferably orally.

Enhancing the Immune System

The compositions of the present invention may be used in the manufactureof a medicament for enhancing the immune system in an animal, preferablya mammal, more preferably livestock and/or racing animals, which mayresult in for example, enhancement (e.g. promotion) of growth and/or anincrease in the efficiency of feed utilisation and/or a generallyincreased well-being (i.e. the overall health of the subject isimproved) in the subject. The overall health of a subject can bedetermined by one or more of the following parameters for example:weight data (with weight gain being a positive determinant), alertness(with full alert being a positive determinant), movement (with energeticmovement as opposed to lethargic movement being a positive determinant)and sickness (with reduced amount of sickness being a positivedeterminant). Typically, the immune modulator composition orpharmaceutical composition according to this aspect of the presentinvention may be an immune enhancer.

Advantageously, the immune modulator composition or pharmaceuticalcomposition of the present invention may be used to replace antibioticsthat are currently used to promote the growth of livestock.

The term “livestock”, as used herein refers to any farmed animal.Preferably, livestock is one or more of poultry (including chickens),pigs (including piglets), sheep (including lambs), cows or bulls(including calves). More preferably, livestock means pigs—includingpiglets.

The present invention also contemplates the genera of the presentinvention being administered in combination with known probioticbacteria, for modification of the cellular immune response.

Commercially at present antibiotics are commonly used as dietaryenhancing feed additives (or growth promoters) and are incorporated intoanimal feed. However, the EU, is expected to introduce a complete ban onthe non-clinical use of antibiotics in animal husbandry. Therefore, themarket requires effective alternatives.

An advantage of the present invention is that it may be used (optionallytogether with good animal husbandry practices) as a replacement todietary enhancing feed additive (or growth promoters).

The immune modulator compositions or pharmaceutical compositions of thepresent invention may be administered as a food additive when used toenhance the immune system.

Cancer

Suitably, the compositions according to the present invention are usedto modulate a cellular immune response to treat and/or prevent cancer.In particular it is envisaged that the compositions according to thepresent invention may be used to protect a subject against developingand/or the progression of a cancer. In particular, the subject with amodulated cellular immune response may be less susceptible to thedevelopment of cancer.

In particular, during cancer growth an unregulated increase in Th2 isobserved.

Cancer is a disease that affects many people, with 65 percent of casesoccurring in those over 65. As the average life expectancy in the UK hasalmost doubled since the mid-nineteenth century, the population at riskof cancer has grown. Death rates from other causes of death, such asheart disease, have fallen in recent years while deaths from cancer haveremained relatively stable. The result is that 1 in 3 people will bediagnosed with cancer during their lifetime and 1 in 4 people will diefrom cancer.

Examples of cancer include bladder, brain tumor, breast cancer, cervicalcancer, colon and rectal cancer, adenocarcinoma, endometrial cancer,esophageal cancer, kidney cancer, leukaemia, liver cancer, lung cancer,melanoma, myeloma, non-hodgkin's lymphoma, ovarian cancer, pancreaticcancer, prostate cancer, sarcoma, soft tissue and stomach cancer.

In addition, persistent smoking of tobacco, and to a lesser, extentpassive smoking, has been associated with carcinomas of the partsdirectly in contact with smoke, oropharynx, trachea, lungs, oesophagusand stomach. As well as these, distant tumours such as those of thekidney, bladder, pancreas, liver and myeloid leukaemia may be increaseby smoking of tobacco. In the present invention, it is envisaged thatcompositions according to the present invention could be administered tosmokers of tobacco in an attempt to reduce the smokers' risk ofdeveloping carcinomas associated with tobacco smoking.

Suitably, the cancer may be an adenocarcinoma or a melanoma.

Suitably, the cancer may be virally related cancers such as cervicalcancer for example. Without wishing to be bound by theory, in someinstances it has been found that an infection caused by papillomaviruses, such as dysplasia of the uterine cervix, precedes carcinoma ofthe cervix. Thus, cervical cancer is herein considered a “virallyrelated cancer”. However, the term “virally related cancer” as usedherein means any cancer which may be caused by or related with a viralinfection.

Post-Operative Recovery, Stress and Infection

Following any major operation a number of situations potentially arise:

Stresses associated with a surgical operation include one or more of thefollowing: apprehension before the operation, stress to the tissues dueto the operative procedures, the pain usually accompanying recovery,worry about the significance of operative findings.

These kinds of stress are associated with the deviation of T-cellfunction towards Th2.

Immunosuppressive effects of premedication and anaesthetics, which maypersist for days or weeks after the operation itself.

For the avoidance of doubt, the term “post-operative stress” as usedherein includes the stress associated with anaesthesia.

In addition, exposure of cut flesh to direct infection at the time ofoperation and of the wound to infection in the recovery room and wardsprior to leaving hospital is also a problem.

A combination of these factors exposes the patient to a series ofpotential bacterial infections, which:

Since the patient is hospitalised, include such notorioushospital-associated infections as those withmethicillin-resistant-Staphylococcus aureus (MRSA). Operations on thebowel expose the patient to gram-negative infections due to exposure ofcut tissues to bowel contents. Operations on the lower limbs are alsosubject to infections with normal members of the gut flora.

Minor infections of the wound delay healing and increase the chances ofcontracting more serious infections.

To counteract these influences, immune regulation towards Th1 and adown-regulation of Th2 a result of the application of the invention,should do one or more of the following: increase non-specific resistanceto post-operative bacterial infections; aid in wound healing and/orreduce stress.

T Helper Cells

The term ‘Th1 ’ as used herein refers to a type 1 T-helper cell (Th1).The term may also be used herein to refer to the response mediated by orthrough such a cell type. Such a response may include one or more of thesecretion of Interleukin-2 (IL-2), the secretion of Interferon-gamma(IFN-γ), activation of macrophages, activation of cytotoxic T-cells, orany other Th1-associated event. Thus, the term ‘Th1 ’ may include Th1cell(s) as well as the immune response(s) which such cell(s) produce.

The term ‘Th2’ as used herein refers to a type 2 T-helper cell (Th2).The term may also be used herein to refer to the response mediated by orthrough such a cell type. Such a response may include one or more of thesecretion of Interleukin-4 (IL-4), the secretion of the splice variantinterleukin IL-4δ2, the secretion of Interleukin-5 (IL-5), increase inlevels of cell determinant 30 (CD30) on lymphocytes, increase in levelsof Immunoglobulin-E (IgE) in the blood or eosinophils in the blood, orany other Th2-associated event. Thus, the term ‘Th2’ may include Th2cell(s) as well as the immune response(s) which such cell(s) produce.

It is known that various conditions may result in or from an unregulatedor inappropriately regulated cellular immune response, in particular inthe activation and/or proliferation of Th1 and/or Th2, which if leftunregulated or inappropriately regulated has been found to result in oneor more detrimental effects on the subject. In particular, such anunregulated or inappropriately regulated cellular immune response hasbeen found to occur following vaccination, e.g. following childhoodvaccinations, and is thought to result in conditions such as the onsetof allergies, i.e. allergic dermatitis and allergic asthma. By way ofexample, Lewis D Curr Opin Immunol 2002; 14: 644 report that Th2 immuneresponses mediated by the secretion of IL-4, IL-5 and IL-13 are key inthe pathogenesis of atopic disorders, including allergen-induced asthma,rhinoconjunctivitis and anaphylaxis. Although such responses aredownregulated to some degree by conventional specific immunotherapy,this approach is only partially effective and has a substantial risk ofadverse effects. Many strategies for immunotherapeutic prophylaxis andfor treatment of atopic diseases have been devised on the basis of mouseallergy models, including the downregulation of Th2 responses by theinduction of regulatory T cell activity, Th2 to Th1 immune deviation,Th1 crossregulation of Th2 immune responses, anergy andimmunosuppressive cytokines. Choi & Koh Ann Allergy Asthma Immunol 2002;88: 584-91 examined whether BCG vaccination of adult patients withasthma, a Th2-associated allergic disease, is clinically effective. Itwas shown that BCG vaccination improved lung function and reducedmedication use in adults with moderate-to-severe asthma. Thisamelioration was accompanied by a suppressed Th2-type immune response,suggesting that BCG vaccination might be an effective therapeuticmodality against asthma. von Hertzen J Allergy Clin Immunol 2002; 109:923-8 outlined the possibility that prolonged maternal stress associatedwith sustained excessive cortisol secretion could affect the developingimmune system—especially Th1/Th2 cell differentiation which may furtherincrease the susceptibility to asthma and atopy in geneticallypredisposed individuals.

In addition, an unregulated or inappropriately regulated cellular immuneresponse has been observed during disease progression. In particularduring cancer growth an unregulated increase in Th2 is observed. By wayof example, Maraveyas et al. Ann Oncol 1999; 10: 817-24 have studied theefficacy of SRL 172 vaccine in patients with cancer i.e. advanced stage1V (AJCC) malignant melanoma. Induction of intracellular cytokines (IL-2and INF-gamma) in peripheral blood lymphocytes (PBLCs) from thesepatients was assayed and correlated to clinical outcome. It wasdemonstrated that SRL 172 was effective in inducing intracellular IL-2responses in a significant number of patients with stage 1V (AJCC)melanoma. Stanford et al. International Journal of PharmaceuticalMedicine 1999; 13: 191-195 report that there is increasing evidence thateffective anti-tumour immune responses are likely to be mediated by type1 cytokines. Recent investigations indicate that heat-killedMycobacterium vaccae, is a reliable Th1 adjuvant and preliminaryclinical trials indicate beneficial effects in melanoma, and cancer ofthe prostate and lung. More extensive controlled studies are currentlybeing conducted to confirm these findings.

An unregulated or inappropriately regulated cellular immune response hasalso been observed during infection and particularly chronic infection,for example during progressive tuberculosis, lepromatous leprosy,visceral leishmaniasis and HIV infection and during allergies. By way ofexample, Clerici & Shearer G M Immunol Today 1993; 14: 107-11 proposethat a Th1 to Th2 switch is a critical step in the etiology of HIVinfection. Clerici & Shearer Immunol Lett 1996; 51: 69-73 show thatHIV-specific cell mediated immunity may be the main correlator ofprotection against HIV infection and against the progression of HIVinfection to AIDS. Abbot N C et al. European Journal of Vascular andEndovascular Surgery 2002 24:202-8 evaluated immunotherapy as a means ofimproving peripheral blood flow in chronic leprosy patients byadministration of heat-killed Mycobacterium vaccae. It was shown thatimmunotherapy, given 18 months earlier, significantly improved bloodflow and temperature sensation, in fully-treated, chronic, leprosypatients.

Accordingly, an aim of the present invention is to promote and establishthe regulation of a cellular immune response, including the regulationor modulation of Th1 and/or Th2, in such a way so as to overcome thenegative effects of the unregulated or inappropriately regulatedcellular immune response.

Suitably, the use of an immune modulator composition and/orpharmaceutical composition according to the present invention modulatesthe Th1 or Th2 response, i.e. a Th1 or Th2 response that results in, forexample, tissue damage.

An unregulated or inappropriately regulated immune response may play arole in the establishment of disease due to the fact that some diseasescause shifted Th1 and/or Th2 responses. Accompanying these atypical Th1and Th2 reactions are a series of abnormal inflammatory responses, whichmay take part in the mechanisms underlying tissue pathology.

By way of example only, the immune modulator composition and/orpharmaceutical composition according to the present invention maycounteract the disadvantages of reduced contact with environmentalinfluences (for example, antigens) commensurate with modern life, maycounteract the influence of treatment of an infection (e.g. a parasiticinfection, such as, for example, malaria, trypanosomiasis,leishmaniasis, and toxoplasmosis) and/or the immunological abnormalitiesaccompanying an infection, stress, such as, for example, major traumastress, psychosocial stress and chronic stress, an allergy (e.g. asthmaincluding asthma, allergic asthma, hayfever, allergic dermatitis(eczema), anaphylactic shock, allergies to plant contact or ingestion,stings—such as nettle and insect stings, and allergies to insectbites—such as midges for instance Culicoides(which causes Sweet Itch inhorses), heaves, COPD and cancer (for example melanoma oradenocarcinoma); an immune system imbalance (e.g. an immune systemimbalance in children e.g. the undesirable effect of childhood vaccinesand the elderly); and post-operative stress and post-operativeinfection.

Vaccines

The preparation of vaccines which contain one or more substances as anactive ingredient(s), is known to one skilled in the art. Typically,such vaccines are prepared as injectables, either as liquid solutions orsuspensions; solid forms suitable for solution in, or suspension in,liquid prior to injection may also be prepared. The preparation may alsobe emulsified, or the active ingredient(s) encapsulated in liposomes.The active ingredients are often mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredient.Suitable excipients are, for example, water, saline, dextrose, glycerol,ethanol, or the like and combinations thereof. Alternatively, thevaccine may be prepared, for example, to be orally ingested and/orcapable of inhalation.

In addition, if desired, the vaccine may contain minor amounts ofauxiliary substances such as wetting or emulsifying agents and pHbuffering agents.

Administration

Typically, a physician will determine the actual dosage of a vaccine,immune modulator composition and pharmaceutical composition which willbe most suitable for an individual subject and it will vary with theage, weight and response of the particular patient. The dosages beloware exemplary of the average case. There can, of course, be individualinstances where higher or lower dosage ranges are merited.

Preferably, the actual dosage that is used results in minimal toxicityto the subject.

The compositions of the present invention may be administered by directinjection. The composition may be formulated for parenteral, mucosal,intramuscular, intravenous, subcutaneous, intraocular, intradermal ortransdermal administration.

Suitably, the composition according to the present invention may beadministered at a dose of 10³-10¹¹ organisms, preferably 10⁴-10¹⁰organisms, more preferably 10⁶-10-5×10⁹ organisms, and even morepreferably 10⁶-10⁹ organisms. Typically, the composition according tothe present invention may be administered at a dose of 10⁸-10⁹ bacteriafor human and animal use.

If the compositions of the present invention are to be administrated asimmune enhancers, then 10³-10¹¹ organisms per dose, preferably 10⁴-10¹⁰organisms per dose, more preferably 10⁶-10-5×10⁹ organisms per dose, andeven more preferably 10⁶-10⁹ organisms per dose, and even morepreferably, 10⁸-10⁹ bacteria per dose for human and animal use may beadministered at regular intervals.

As will be readily appreciated by a skilled person the dosageadministered will be dependent upon the organism to which the dose isbeing administered.

The term “administered” includes delivery by delivery mechanismsincluding injection, lipid mediated transfection, liposomes,immunoliposomes, lipofectin, cationic facial amphiphiles (CFAs) andcombinations thereof, or even viral delivery. The routes for suchdelivery mechanisms include but are not limited to mucosal, nasal, oral,parenteral, gastrointestinal, topical, or sublingual routes.

The term “administered” includes but is not limited to delivery by amucosal route, for example, as a nasal spray or aerosol for inhalationor as an ingestable solution; a parenteral route where delivery is by aninjectable form, such as, for example, an intravenous, intramuscular,intradermal or subcutaneous route.

The term “co-administered” means that the site and time ofadministration of each of the adjuvants(s), antigen(s) and/or antigenicdeterminant(s) of the present invention are such that the necessarymodulation of the immune system is achieved. Thus, whilst the antigen(s)and adjuvant(s) may be administered at the same moment in time and atthe same site, there may be advantages in administering the antigen(s)and/or antigenic determinant(s) at a different time and to a differentsite from the adjuvant(s). The antigen(s) and/or antigenicdeterminant(s) and adjuvant(s) may even be delivered in the samedelivery vehicle—and the antigen(s) and/or antigenic determinant(s) andadjuvant(s) may be coupled and/or uncoupled and/or genetically coupledand/or uncoupled. By way of example only, the immune modulatorcomposition according to the present invention may be administeredbefore, at the same time or post administration of one or more antigensor further antigens.

The antigen, antigenic determinant, peptide or homologue or mimeticthereof may be administered separately or co-administered to the hostsubject as a single dose or in multiple doses.

The immune modulator composition and/or pharmaceutical composition ofthe invention may be administered by a number of different routes suchas injection (which includes parenteral, subcutaneous, intradermal andintramuscular injection) intranasal, mucosal, oral, intra-vaginal,urethral or ocular administration.

Preferably, in the present invention, administration is by injection.More preferably the injection is intradermal.

Preferably, in the present invention, administration is by an orallyacceptable composition.

For vaccination the composition can be provided in 0.1 to 0.2 ml ofaqueous solution, preferably buffered physiological saline, andadministered parenterally, for example by intradermal inoculation. Thevaccine according to the invention is preferably injected intradermally.Slight swelling and redness, sometimes also itching may be found at theinjection site. The mode of administration, the dose and the number ofadministrations can be optimised by those skilled in the art in a knownmanner.

Antigens

As used herein, an “antigen” means an entity which, when introduced intoan immunocompetent host, modifies the production of a specific antibodyor antibodies that can combine with the entity, and/or modifies therelevant Th response, such as Th2 and/or Th1. The antigen may be a puresubstance, a mixture of substances or soluble or particulate material(including cells or cell fragments or cell sonicate). In this sense, theterm includes any suitable antigenic determinant, cross reactingantigen, alloantigen, xenoantigen, tolerogen, allergen, hapten, andimmunogen, or parts thereof, as well as any combination thereof, andthese terms are used interchangeably throughout the text.

The term “antigenic determinant or epitope” as used herein refers to asite on an antigen which is recognised by an antibody or T-cellreceptor, or is responsible for evoking the T-helper cell response.Preferably it is a short peptide derived from or as part of a proteinantigen. However the term is also intended to include glycopeptides andcarbohydrate epitopes. The term also includes modified sequences ofamino acids or carbohydrates which stimulate responses which recognisethe whole organism.

It is advantageous if the antigenic determinant is an antigenicdeterminant of the infectious agent which causes the infectious disease.

A “preventative” or “prophylactic” vaccine is a vaccine which isadministered to naive individuals to prevent development of a condition,such as by stimulating protective immunity.

A “therapeutic” vaccine is a vaccine which is administered toindividuals with an existing condition to reduce or minimise thecondition or to abrogate the immunopathological consequences of thecondition.

Adjuvants

The term ‘adjuvant’ as used herein means an entity capable of augmentingor participating in the influencing of an immune response. An adjuvantis any substance or mixture of substances that assists, increases,downregulates, modifies or diversifies the immune response to anantigen.

The immune modulator composition and/or pharmaceutical compositionaccording to the present invention may comprise one or more adjuvantswhich enhance the effectiveness of the immune modulator compositionand/or pharmaceutical compositions. Examples of additional adjuvantswhich, may be effective include but are not limited to: whole cells of abacterium from one or more of the following genera Tsukamurella,Rhodococcus, Gordonia, Nocardia, Dietzia, Mycobacterium, aluminiumhydroxide, aluminium phosphate, aluminium potassium sulphate (alum),beryllium sulphate, silica, kaolin, carbon, water-in-oil emulsions,oil-in-water emulsions, muramyl dipeptide, bacterial endotoxin, lipid X,Corynebacterium parvum (Propionobacterium acnes), Bordetella pertussis,Mycobacterium vaccae, Mycobacteium obuense, polyribonucleotides, sodiumalginate, lanolin, lysolecithin, vitamin A, interleukins such asinterleukin 2 and interleukin-12, saponin, liposomes, levamisole,DEAE-dextran, blocked copolymers or other synthetic adjuvants. Suchadjuvants are available commercially from various sources, for example,Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.) or Freund'sIncomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit,Mich.). Only aluminium hydroxide is approved for human use. Some of theother adjuvants, such as M. vaccae for example, have been approved forclinical trials.

Suitably, the adjuvant may be a whole cell of a bacterium from a roughstrain of Mycobacterium obuense.

In the art, it is known that DNA vaccines, which are essentially DNAsequences attached to gold particles and which are fired into the skinby a helium gun, are efficient vaccine delivery systems. Unlikeconventional vaccines, these DNA vaccines do not require a traditionaladjuvant component. In accordance with a further aspect of the presentinvention, the immune modulator composition as defined herein maysuitably be used in conjunction with such DNA vaccines to augment orparticipate in the influencing of the immune response.

Pharmaceutical Compositions

The present invention also provides a pharmaceutical compositioncomprising a therapeutically effective amount of a whole cell of abacterium from a rough strain of Mycobacterium obuense and optionally apharmaceutically acceptable carrier, diluent or excipients (includingcombinations thereof).

The pharmaceutical composition may comprise two components—a firstcomponent comprising an antigen and a second component comprising anadjuvant thereof. The first and second component may be deliveredsequentially, simultaneously or together, and even by differentadministration routes.

Suitably, the antigen may even be engendered within the host tissues aspart of a disease process. Thus, antigen may originate from a bacterial,host or parasitic invasion, or may be a substance release from thetissues such as a stress protein or a tumour antigen.

The pharmaceutical compositions may be for human or animal usage inhuman and veterinary medicine and will typically comprise any one ormore of a pharmaceutically acceptable diluent, carrier, or excipient.Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).The choice of pharmaceutical carrier, excipient or diluent can beselected with regard to the intended route of administration andstandard pharmaceutical practice. The pharmaceutical compositions maycomprise as—or in addition to—the carrier, excipient or diluent anysuitable binder(s), lubricant(s), suspending agent(s), coating agent(s),solubilising agent(s).

Preservatives, stabilisers, dyes and even flavouring agents may beprovided in the pharmaceutical composition. Examples of preservativesinclude sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid. Antioxidants and suspending agents may be also used.

There may be different composition/formulation requirements dependent onthe different delivery systems. By way of example, the pharmaceuticalcomposition of the present invention may be formulated to be deliveredusing a mini-pump or by a mucosal route, for example, as a nasal sprayor aerosol for inhalation or ingestable solution, or parenterally inwhich the composition is formulated by an injectable form, for delivery,by, for example, an intravenous, intramuscular, intradermal orsubcutaneous route. Alternatively, the formulation may be designed to bedelivered by both routes.

Preferably in the present invention the formulation is of injectableform. More preferably the formulation is intradermally injected.

Preferably in the present invention the formulation is an orallyacceptable composition.

Where the agent is to be delivered mucosally through thegastrointestinal mucosa, it should be able to remain stable duringtransit through the gastrointestinal tract; for example, it should beresistant to proteolytic degradation, stable at acid pH and resistant tothe detergent effects of bile.

Where appropriate, the pharmaceutical compositions can be administeredby inhalation, in the form of a suppository or pessary, topically in theform of a lotion, solution, cream, ointment or dusting powder, by use ofa skin patch, orally in the form of tablets containing excipients suchas starch or lactose, or in capsules or ovules either alone or inadmixture with excipients, or in the form of elixirs, solutions orsuspensions containing flavouring or colouring agents, or they can beinjected parenterally, for example intravenously, intramuscularly,intradermally or subcutaneously. For parenteral administration, thecompositions may be best used in the form of a sterile aqueous solutionwhich may contain other substances, for example enough salts ormonosaccharides to make the solution isotonic with blood. For buccal orsublingual administration the compositions may be administered in theform of tablets or lozenges which can be formulated in a conventionalmanner.

Pharmaceutical Combinations

The agent of the present invention may be administered with one or moreother pharmaceutically active substances. By way of example, the presentinvention covers the simultaneous, or sequential treatments with animmune modulator composition and/or pharmaceutical composition accordingto the present invention, and one or more steroids, analgesics,antivirals, interleukins such as IL-2, or other pharmaceutically activesubstance(s).

It will be understood that these regimes include the administration ofthe substances sequentially, simultaneously or together.

Immune Enhancer

The term “immune enhancer” as used herein means one or more bacteriaeither isolated or in culture which when administered to a subjectbenefit the health of that subject. Preferably, this benefit is achievedby the modification of the cellular immune response of the subject.

In accordance with the present invention, immune enhancers may be used,for example, for the treatment or prevention of an immune systemimbalance in a subject, preferably a child or an elderly subject, or forenhancing the immune system of a subject, for example of a mammal,particularly of livestock or of humans.

The immune enhancers may be administered by consumption in speciallydesigned food or in animal feeds, for example pig animal feedssupplemented with the bacteria of the present invention.

The immune enhancers may also be administered by other routes—such asdirect injection.

Preferably, the bacteria are killed so as to avoid the difficulties ofmaintaining live products.

Identifying a Bacterium that Modulates a Cellular Immune Response

In another aspect, the present invention relates to a method foridentifying one or more whole cells of bacteria from a rough strain ofMycobacterium that modulate (e.g. modify) a cellular immune responsecomprising the steps of: (a) contacting a first test animal with animmunostimulant; (b) contacting a second test animal with animmunostimulant mixed with a bacterium; (c) measuring the cellularimmune response in each of the test animals; and (d) comparing thecellular immune response in each of the test animals, wherein, a lowercellular immune response from the immunostimulant mixed with a bacteriumin comparison to the immunostimulant alone is indicative of amodification of the cellular immune response by the bacterium.

In another aspect, the present invention relates to a method ofdetermining the Th1/Th2 response of a rough strain of bacteria selectedfrom the genus Mycobacterium which method comprises utilisation of thetuberculin skin test. In mice, the tuberculin skin test is preferablycarried out on the foot pad. In a predominant Th1 reaction the positivefoot pad immune response is maximal at 24 hours and diminishes at 48hours. However, as the Th2 reactivity increases then the 48 hourpositive foot pad immune response increases and can even exceed the footpad immune response at 24 hour.

The effect of BCG vaccination is well documented using this tuberculinskin test. Thus, the test assay can be used to assess whether or not theintroduction of an immune modulator composition according to the presentinvention modulates the BCG cellular immune response.

As used herein, the term “test animal” refers to any animal that elicitsa cellular immune response to the immunostimulant. Preferably, the testanimal(s) is a mammal. More preferably, the test animal(s) is a rat,hamster, rabbit, guinea pig or mouse. More preferably, the testanimal(s) is a mouse.

Preferably, the bacterium modifies the T helper cell response. Suitably,the bacterium may modify the T helper cell response by decreasing theTh1 and Th2 response. Suitably, the bacterium may modify the T helpercell response by increasing the Th1 response and decreasing the Th2response. Suitably, the bacterium may modify the T helper cell responseby increasing the Th1 response without affecting the Th2 response.

Preferably, the immunostimulant will have a known Th1 and Th2 response.For example, with the immunostimulant BCG the reaction is usuallylargest at 24 h when it is an indicator of the Th1 response; thereaction at 48 h is usually less and includes a Th2 contribution. It isknown that BCG predominantly stimulates a Th1 response.

By use of such immunostimulants it may be possible to determine theTh1/Th2 response of a test bacterium and, thus, it may be possible toidentify one or more bacteria which have a desired Th1/Th2 response totreat and/or prevent a particular disease and/or disorder.

Preferably, the cellular immune response is measured using thetuberculin skin test. Vaccination with an immunostimulant—such asBCG—induces a response to skin-testing with tuberculin (a solublepreparation of Tubercle bacilli), when tested later. The local reactionis measured at various intervals, for example, 24 hours, 48 hours and 72hours after injection of tuberculin. Briefly, an immunostimulant (e.g.BCG) is used that induces a positive immune response to tuberculin. Inthe test animal, the tuberculin skin test is preferably carried out onthe foot pad. In a predominant Th1 reaction the positive foot pad immuneresponse is usually maximal at 24 hours and diminishes at 48 hours.However, as the Th2 reactivity increases then the 48 hour positive footpad immune response increases and can even exceed the foot pad immuneresponse at 24 hour. Thus, the assay can be used to assess whether ornot the introduction of an immune modulator composition according to thepresent invention modulates the cellular immune response.

Preferably, the immunostimulant is BCG.

The invention will now be further described by way of Examples, whichare meant to serve to assist one of ordinary skill in the art incarrying out the invention and are not intended in any way to limit thescope of the invention.

EXAMPLES Methods Tuberculin Skin Test

The tuberculin skin test is an appropriate model assay to assess theeffect of an immune modulator composition, i.e. bacterialcompositions/suspensions comprising whole killed bacterial cellsaccording to the present invention, on a cellular immune response.

BCG vaccination induces a positive immune response to tuberculin. Inmice, the tuberculin skin test is preferably carried out on the footpad. In a predominant Th1 reaction the positive foot pad immune responseis maximal at 24 hours and diminishes at 48 hours. However, as the Th2reactivity increases then the 48 hour positive foot pad immune responseincreases and can even exceed the foot pad immune response at 24 hour.

The effect of BCG vaccination is well documented using this tuberculinskin test. Thus, the test assay can be used to assess whether or not theintroduction of an immune modulator composition according to the presentinvention modulates the BCG cellular immune response.

Preparation of a Bacterial Suspension

The bacteria of a rough strain from the genus Mycobacteria may be grownin an antigen-free medium, such as Sauton's medium, in a fermenter for2-28 days. Alternatively, the bacterial species of interest may be grownon a solid slope. Alternative methods would be readily available tothose skilled in the art.

The resulting bacterial mass may be harvested and either used directlyor after washing to make a suspension in buffer. The bacterial cellsuspension is prepared to contain between 100,000 and 10,000,000,000bacilli per dose. The bacterial cells are resuspended in water or in asaline. Preferably, the saline is buffered at pH 8.0 with borate.Preferably the bacilli are inactivated (killed), suitably by heating inan autoclave for 15 minutes at 121° C. The resulting bacterialsuspension comprises whole cells.

Example 1 Induction of a Stable Rough Variant of Mycobacterium obuenseType Strain (ATCC 27023)

Mycobacterium obuense type strain ATCC 27023 is only represented by thisstrain in the international collections. The strain is smooth incultural character and is not known to throw rough variants.

Experiment 1. Culture on to Middlebrook 7H11 Agar.

Six consecutive subcultures of Mycobacterium obuense type strain ATCC27023 onto Middlebrook 7H11 agar produced smooth colonies only.

Experiment 2. Culture on to Middlebrook 7H11 Agar with DifferentConcentrations of Added 4,4′-Diaminodiphenyl Sulfone

Experiment 2, Part a

Mycobacterium obuense type strain ATCC 27023 was grown Middlebrook 7H11medium comprising different concentrations of Dapsone™. Results arerecorded in Table 1.

TABLE 1 Growth on different Dapsone ™ concentrations/ml of Middlebrook7H11 medium. μg Dapsone ™/ml Middlebrook 7H11 medium Observed growth 50no growth 40 no growth 20 no growth 10 no growth 5 3 smooth colonies 0confluent smooth growth

Experiment 2, Part b

Mycobacterium obuense type strain ATCC 27023 was grown Middlebrook 7H11medium comprising different concentrations of Dapsone™. Results arerecorded in Table 2.

TABLE 2 Growth on different Dapsone ™ concentrations/ml of Middlebrook7H11 medium. μg Dapsone ™/ml Middlebrook 7H11 medium Observed growth 5small smooth colonies 4 small smooth colonies 3 confluent smoothcolonies 0 confluent smooth coloniesExperiment 2, Part c Mycobacterium obuense type strain ATCC 27023 wasgrown Middlebrook 7H11 medium comprising different concentrations ofDapsone™. Results are recorded in Table 3.

TABLE 3 Growth on different Dapsone ™ concentrations/ml of Middlebrook7H11 medium. μg Dapsone ™/ml Middlebrook 7H11 medium Observed growth 12mostly rough colonies 10 mostly rough colonies 7.5 few rough colonies,mostly smooth colonies 5 few rough colonies, mostly smooth colonies 0confluent smooth colonies

Rough colonies were carefully picked off from the 12 μg Dapsone™/mlculture and subcultured repeatedly on Middlebrook 7H11 medium with noadded Dapsone™

Only rough colonies have grown after 20 repeated subcultures (i.e.successional cultures).

The rough strain isolated using this experiment has been deposited withthe NCTC under the Budapest Convention under accession number NCTC13365.

Example 2 DTH Reactions to Tuberculin in BCG-Challenged Mice which HadBeen Treated with M. Obuense

The experimental group was given, 10⁷ bacilli/0.1 ml of theimmunomodulator, at birth and 21 days later (the time of weaning).Control mice received buffered borate under the same schedule.

Thirty days following the last immunisation mice were vaccinated intothe scruff of the neck with BCG subcutaneously (Merieux) 10⁵ bacilli/0.1ml

Twenty eight days later animals were challenged with tuberculin (righthind footpad) or saline (left hind footpad) 0.02 ml.

Alternatively, the foot pad thickness can be measured before testinginstead of injecting the left hind footpad, as well.

DTH reactions were measured at 24, 48 and 72 h post-challenge

Individual Data

GROUP 24 h 48 h 72 h Borate 0 0 0 Borate 14 3 7 Borate 12 22 25 Borate15 12 13 Borate 4 1 6 Borate 0 0 0 Borate 0 0 0 Borate 10 4 3 Borate 1312 14 M. obuense NCTC 13365 1 1 1 M. obuense NCTC 13365 1 2 2 M. obuenseNCTC 13365 2 6 4 M. obuense NCTC 13365 0 4 2 M. obuense NCTC 13365 5 614 M. obuense NCTC 13365 6 6 9 M. obuense NCTC 13365 0 0 0 M. obuenseNCTC 13365 1 4 4 Hours Borate (n = 9) M. obuense (n = 8) 24 7.5 ± 2.16 2 ± 0.8 48   6 ± 2.57 3.6 ± 0.85 72 7.5 ± 2.8  4.5 ± 1.66 Values aremeans ± sem

There are no significant differences when non-responders and respondersare pooled in this way.

However, when responders alone are analysed, significant differences aredisclosed.

Hours 24 48 72 Borate 11.33 ± 3.98 10.60 ± 7.67 11.33 ± 7.92 Mean + ve n= 6 n = 5 n = 6 response size M. obuense  2.50 ± 2.43  4.67 ± 1.63  5.83± 4.75 Mean + ve n = 6 n = 6 n = 6 response size

Differences in +ve response sizes to tuberculin between theborate-primed control and the results after priming with a rough strainof M. obuense are statistically significant at 24 hours (p<0.005) andalthough significance is lost at 48 and 72 hours, there is a downwardtrend.

A positive response to tuberculin means that effective cells have beendrawn to the site of injection and that inflammatory cytokines have beenreleased. Thus, in a negative response in which cells have beenrecruited to the site, but have not released cytokines, swelling, whichis due chiefly to the response to released cytokines may not beappreciable, even though competent cells have been recruited to thesite.

In an animal that has been immunised with reagents containing antigensshared with the test reagent, a lack of response does not mean thatthere is no response, just that the test has not detected it. Negativeresponse may occur because there is an incapability of responding orthere is a response but in a negative sense. The latter circumstance maybe likely to happen in DTH reactions in BCG-challenged animals. Withoutwishing to be bound by theory, it is believed that sensitised cells mayremain in lymph nodes instead of migrating to the dermis or that theymay get there but produce downregulating cytokines, anergy-inducingcells. Sometimes, an alternative test such as the lymphocytetransformation test can demonstrate the presence of competentcirculating cells responsive to the test antigen, and punch biopsies ofapparently negative skin-test sites will show infiltrations of thesecells, which nonetheless are not producing a perceptible swelling. Theproportion of test-negative individuals, skin- or footpad-tested, varieswith genetics and time scale in relation to priming and challenge. Thereis a large body of published evidence dealing with these phenomena inman.

Thus in analysis of data from such tests, one needs to look atproportion making a positive response and mean diameter of positiveresponses, as we have done. There was no difference in percentresponding between the 2 groups, but there was a difference in the sizesof response that responders made.

CONCLUSIONS

M. obuense (rough strain NCTC 13365) has an immunoregulatory effect.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the present invention will be apparentto those skilled in the art without departing from the scope and spiritof the present invention. Although the present invention has beendescribed in connection with specific preferred embodiments, it shouldbe understood that the invention as claimed should not be unduly limitedto such specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in biochemistry and immunology or related fields areintended to be within the scope of the following claims.

REFERENCES

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1. A method of producing rough strains of a bacterium said methodcomprising exposing said bacterium to 4,4′-Diaminodiphenyl sulfone in anamount which is greater than or equal to 7.5 μg/ml and less than 20μg/ml wherein said bacterium is from an aerobic genus Mycobacterium. 2.A method according to claim 1 wherein said rough strain is isolated. 3.A method according to claim 1 wherein said bacterium is Mycobacteriumobuense.
 4. A rough strain of Mycobacterium obuense producible by themethod according to claim
 1. 5. A rough strain of Mycobacterium obuensedeposited under the Budapest Treaty at the NCTC with the accessionnumber NCTC
 13365. 6. An immune modulator composition comprising a wholecell of a rough strain of Mycobacterium obuense according to claim 4,wherein said immune modulator composition in use modifies a cellularimmune response.
 7. An immune modulator composition according to claim 6wherein said composition comprises an antigen and an adjuvant, whereinsaid adjuvant comprises a whole cell of a rough strain of Mycobacteriumobuense.
 8. A pharmaceutical composition comprising a whole cell of abacterium from a rough strain of Mycobacterium obuense and optionally apharmaceutically acceptable carrier, diluent or excipient, whichpharmaceutical composition in use modifies a cellular immune response.9. Use of an immune modulator composition or a pharmaceuticalcomposition according to claim 6 in the manufacture of a medicament forthe treatment or prevention of one or more of an infection (e.g. abacterial, viral, or parasitic infection, for example, malaria,trypanosomiasis, leishmaniasis, infection with Eimeria species inpoultry, and toxoplasmosis) and/or the immunological abnormalitiesaccompanying an infection, an autoimmune disease (e.g. a vasculardisorder, such as obliterative vascular disorder, and the immunologicalaspects underlying myointimal hyperplasia and/or atheroma formation(otherwise known as arteriosclerosis), arthritis and graft rejection.),stress (for example, major trauma stress, psychosocial stress andchronic stress), an allergy (e.g. asthma including allergic asthma,hayfever, allergic dermatitis (eczema), anaphylactic shock, allergies toplant contact or ingestion, stings—such as nettle and insect stings, andallergies to insect bites—such as midges, for instance Culicoides(whichcauses Sweet Itch in horses)), heaves, COPD and cancer (for examplemelanoma or adenoma-carcinoma) and an immune system imbalance (e.g. animmune system imbalance in children and the elderly).
 10. Use of animmune modulator composition or a pharmaceutical composition accordingto claim 6 in the manufacture of a medicament for the treatment orprevention of one or more of an infection, an autoimmune disease,stress, an allergy, heaves, COPD, cancer and an immune system imbalance.11. Use of an immune modulator composition or a pharmaceuticalcomposition according claim 6 in the manufacture of a medicament for thetreatment or prevention of an adverse reaction to childhood vaccinesand/or consequences thereof.
 12. Use of an immune modulator compositionor a pharmaceutical composition according to claim 6 in the manufactureof a medicament for modifying a cellular immune response.
 13. An immunemodulator composition or a pharmaceutical composition according claim 6for use as a medicament.
 14. An immune modulator composition or apharmaceutical composition according claim 6 for use in or as a vaccine.15. An immune modulator composition according to claim 14 wherein saidvaccine is a prophylactic vaccine or a therapeutic vaccine.
 16. Animmune modulator composition according to claim 14 wherein saidcomposition modifies a cellular immune response.
 17. An immune modulatorcomposition and/or a pharmaceutical composition according claim 6wherein said composition further comprises an antigen or antigenicdeterminant.
 18. An immune modulator composition and/or a pharmaceuticalcomposition according to claim 17 wherein said antigen or antigenicdeterminant is an antigen or antigenic determinant selected from one ormore of a BCG (bacillus of Calmette and Guerin) vaccine, a diphtheriatoxoid vaccine, a diphtheria/tetanus/pertussis vaccine, a pertussisvaccine, the tetanus toxoid vaccine, the measles vaccine, the mumpsvaccine, the rubella vaccine, the OPV (oral poliomyelitis vaccine) andMycobacterium vaccae, or part thereof.
 19. An immune modulatorcomposition and/or pharmaceutical composition according to claim 17wherein said composition comprises two or more such antigens orantigenic determinants.
 20. An immune modulator composition and/orpharmaceutical composition according to claim 6 wherein said bacteriumis killed.
 21. A method for treating or preventing a condition in asubject comprising administering an effective amount of a pharmaceuticalcomposition and/or immune modulator composition according to claim 6 toa subject wherein said composition modulates a cellular immune response.22. A method for immunising a subject comprising administering apharmaceutical composition and/or immune modulator composition accordingto claim
 6. 23. A method according to claim 21 wherein saidpharmaceutical composition and/or an immune modulator composition isco-administered with an antigen or antigenic determinant.
 24. A methodaccording to claim 23 wherein the antigen or antigenic determinant is anantigen or antigenic determinant selected from one or more of a BCG(bacillus of Calmette and Guerin) vaccine, a diphtheria toxoid vaccine,a diphtheria/tetanus/pertussis vaccine, a pertussis vaccine, a tetanustoxoid vaccine, a measles vaccine, a mumps vaccine, a rubella vaccine, aOPV (oral poliomyelitis vaccine) and Mycobacterium vaccae, or partthereof.
 25. A method according to claim 23 wherein said pharmaceuticalcomposition and/or an immune modulator composition is co-administeredwith two or more such antigens or antigenic determinants. 26-29.(canceled)